Relative expression analysis of MYB94 under abiotic stress
The relative expression analysis was performed to determine the MYB94 gene in two groundnut cultivars subjected to drought and salt stress. The results showed that changes in MYB94 gene expression levels varied significantly between two cultivars in response to drought and salt stress. MYB94 transcripts were found to be significantly increased in two groundnut cultivars. The relative expression was 1.3 and 4.3-fold in cultivar K-6 and K-9, respectively, in drought-stressed conditions, whereas it was a 1.5- and 4.8-fold increase in cultivar K-6 and K-9, respectively, in salt-stressed conditions. The results also revealed that the cultivar K-6 showed a less transcript abundance of the MYB94 gene. In contrast, cultivar K-9 showed higher transcript abundance than other cultivars under drought and salt stress (Fig. 1a). Based on the results, MYB94 gene was upregulated more in stress-tolerant cultivar K-9 than cultivar K-6 due to salt treatments.
Isolation and bioinformatic analysis of full-length AhMYB94 gene
The full-length MYB94 gene was isolated from groundnut cultivar K-9 and designated AhMYB94 (GenBank Accession: #MW473683). The AhMYB94 gene sequence contains 921bp nucleotide length, and the AhMYB94 gene-encoded protein contains 306 amino acids with a predicted molecular weight of 34.90 kDa and an isoelectric point (pI) of 6.46. The AhMYB94 protein conserved domain analysis revealed the presence of two MYB DNA binding domains from 14 to 61 and 67 to 112 of 46 amino acid residues. The phylogenetic tree was showed the relationship of MYB94 from Arachis hypogaea with other species MYB related transcription factor genes. The phylogenetic analysis displayed that the MYB94 from Arachis hypogaea is closely related to Arachis hypogaea Transcription factor MYB94 (GenBank: XM 025762415.1) and predicted: Arachis ipaensis related protein 306 (GenBank: XM 016321205) (Fig. 1b). The multiple sequence alignment and Circos plot of the AhMYB94 gene with other MYB94 genes indicated that the homology of AhMYB94 and those MYB94 proteins from other plant species are significantly different except at conserved MYB domains region (Supplementary Fig. 5).
Vector Construction and Transformation of AhMYB94 in groundnut (cultivar K-6)
The AhMYB94 gene was amplified with the gene-specific primers, and the product was recovered by restriction digestion with SacI and XbaI. The cloned TA (pTZ57R/T) plasmid was digested with SacI and XbaI restriction enzymes and ligated into a plant expression vector, pRT101, to obtain CaMV35S constitutive promoter and a polyA terminator. In the next step, the entire gene from pRT101 was digested with Hind III restriction enzyme and cloned into plant binary vector pCAMBIA2301. Restriction and PCR analysis are verified the binary vector pCAMBIA 2301 (pCAMBIA2301:35S:NptII: AhMYB94:GUS) (Fig. 2a), which was transferred into Agrobacterium strain EHA105. A tissue culture-independent in planta transformation technique was used to transfer the AhMYB94 gene into groundnut cultivar K-6 seedlings (Rohini and Rao 2001). Kanamycin resistance was tested in the transformants of T1 plants. Due to the transgene construct, putative transformants were resistant to kanamycin (200 mg/L), and seedlings showed normal growth, whereas WT and mock plants did not show any growth (Fig. 2b). Only healthy and resistant plants were picked and advanced to the next transgenic generation.
Molecular analysis of Transgenic groundnut plants
PCR was carried out to screen the presence of the transgene in genomic DNA isolated from leaves of WT, mock, AhMYB94 transgenic groundnut plants using neomycin phosphotransferase gene, a selectable marker gene (NptII) primer, and β-Glucuronidase a reporter gene (GUS) primers and gene-specific primers. In each generation, the putative transformants showed NptII and GUS integration with 500 bp fragments and 921 bp of AhMYB94 with gene-specific primers. In contrast, wild-type plants showed no amplification with NptII and GUS primers.
Histochemical localization of reactive oxygen species (O2●− and H2O2)
Histochemical localization was employed to bring out in situ accumulation of reactive oxygen species (O2●− and H2O2) in WT, mock and AhMYB94 transgenic groundnut plants. The results show that when WT, mock, and AhMYB94 transgenic groundnut plants were exposed to salt stress, they accumulated O2●− and H2O2 free radicals. ROS accumulation differed significantly between WT, mock, and AhMYB94 transgenic groundnut plants. The production of purple (DAB) and brown (NBT) spots, which indicate superoxide and hydrogen peroxide accumulation, was more abundant in wild-type and mock plants; however, the transgenic groundnut plants had less localized spots (Fig. 2c).
Physio-Biochemical analysis of AhMYB94 transgenic groundnut plants under salt stress
One-month-old plants were subjected to salt stress to analyze stress-induced changes, such as CMS, MDA, O2●− content; H2O2 content, APX and SOD activity, free proline, glycine betaine, and soluble sugar content, for 7-days and all physio-biochemical analyses, were conducted on leaf samples of WT, mock and AhMYB94 transgenic groundnut plants.
Cell membrane stability
The cell membrane stability (% electrolyte leakage) was estimated in WT, mock, and AhMYB94 transgenic groundnut plants under salt stress conditions. The percentage of electrolyte leakage in WT and mock plants was (16.6 and 16.53) significantly higher than transgenic groundnut plants (8.72 – 10.41%) under salt stress (Fig. 3a).
Malondialdehyde (MDA) content
Malondialdehyde (MDA) is a byproduct of unsaturated fatty acid peroxidation in phospholipids and is an indicator of membrane damage. Leaf MDA content was estimated in WT, mock, and AhMYB94 transgenic groundnut lines under salt stress conditions. The membrane damage in the leaves of WT and mock plants was (13.25 and 13.13 nmol/g FW) higher than AhMYB94 transgenic groundnut lines (7.43 – 8.64 nmol/g FW) under salt stress (Fig. 3b).
Superoxide (O2●−) ion and Hydrogen peroxide (H2O2) content
Under salt stress, the synthesis and degradation of ROS were analyzed in WT, mock and AhMYB94 transgenic groundnut plants. ROS such as O2●− ion and H2O2 is produced due to stress-induced oxidative damage. The oxidative damage was more significant in WT and mock leaves than in the AhMYB94 transgenic groundnut plants under salt stress (Fig. 4a,b).
Activities of enzymatic antioxidants
The enzymatic antioxidants such as SOD and APX activity were assayed in the leaves of WT, mock and AhMYB94 transgenic groundnut plants under salt stress. The SOD and APX help to scavenge potentially harmful oxygen molecules in cells. Both SOD and APX activities were significantly increased in wild type, mock, and transgenic groundnut plants. Nevertheless, the increase was more in AhMYB94 transgenic groundnut plants than in the WT and mock plants (Fig. 4c,d).
Total free proline content
Proline is a multifunctional amino acid that can play a significant role in response to biotic and abiotic stress. Free proline accumulation was estimated in the leaves of WT, mock and AhMYB94 transgenic groundnut plants under salt stress. The free proline content was wild-type (65.09 µg/g FW) plants and Mock (68.87 µg/g FW) plants lower than that of transgenic groundnut (136.5 – 153.50 µg/g FW) during salt stress (Fig. 5a).
Glycine betaine (GB) content
Glycine betaine is a common compatible solute, which plays an essential role in response to abiotic stress. GB content was estimated in WT, mock and AhMYB94 transgenic groundnut plants under salt stress conditions. The glycine betaine content was increased in all the groundnut plants under salt stress. However, the WT and mock plants showed lower glycine betaine content than AhMYB94 transgenic groundnut plants. The GB content was 63.45 µg/mg FW in wild type and 69.07 µg/mg FW in mock plants and 139.14 – 151.35 µg/mg FW in transgenic groundnut plants, respectively (Fig. 5b).
Total soluble sugar content
In plants, the soluble sugars protect the cell from osmolysis and are also involved in maintaining ROS balance under stress conditions. Under salt stress conditions, the total soluble sugar content was estimated in WT, mock and AhMYB94 transgenic groundnut plants. The soluble sugars content increased in all the groundnut plants, but the transgenic groundnut plants showed a more percent increase in soluble sugars content than WT and mock plants. The soluble sugars content was 311.95 µg/g FW in wild-type plants and 350.07 µg/g FW in mock plants and 778.8 – 931.63 µg/g FW in transgenic groundnut plants, respectively (Fig. 5c).
Ion content (Na+, K+, and Ca2+ ) analysis of transgenic groundnut plants
The ion contents were quantified in roots and leaves of WT, mock and transgenic groundnut plants under salt stress conditions. The Na+ content in roots of WT mock plants was lesser than transgenic groundnut plants. In contrast, leaf tissues of transgenic plants showed lesser Na+ content when compared to WT and mock plants under salt stress conditions (Fig. 6a). The K+ content in roots of wild type, mock plants exhibited lesser than transgenic groundnut plants and in leaf tissues of WT and mock plants shows lower than that of AhMYB94 transgenic groundnut plants under salt stress (Fig. 6b). In roots, transgenic groundnut plants showed higher calcium ion content than WT and mock plants. In leaf tissues, calcium content was lesser in transgenic groundnut plants than in mock and WT plants under salt stress conditions (Fig. 6c).
Relative expression analysis of AhMYB94 gene in transgenic groundnut plants
In terms of transcript abundance, the relative expression of the AhMYB94 gene was measured as fold change by using SYBR Green-based qRT-PCR analysis in WT, mock, and AhMYB94 transgenic groundnut plants. According to the findings, the transcript levels of the AhMYB94 gene were increased by ̴ three to four folds in transgenic groundnut plants compared to WT and mock plants under salt stress (Fig. 7a).
Relative expression of stress-responsive genes in transgenic groundnut plants
To better understand the involvement of the MYB TF in the mechanism of salt tolerance, the seven downstream stress-responsive genes were selected and examined the relative expression in WT, mock and AhMYB94 transgenic groundnut plants under salt stress conditions. An effective antioxidant system is required to mitigate the oxidative damage produced by salt stress. The relative expression of ROS-scavenging enzymes (SOD, APX, and CAT genes) and other salt stress-responsive genes like NHX1, SOS1, BADH, GSMT were checked using qRT-PCR. Salt stress enhanced expression levels of stress-responsive genes, such as NHX1, SOS1, BADH, GSMT, SOD, APX, CAT in transgenic groundnut plants compared to WT and mock plants (Fig. 7b). Overall, these findings imply that AhMYB94 overexpression regulates the expression of downstream stress-responsive genes under salt stress conditions.
Leaf Senescence assay
The leaf disc senescence assay was used to investigate the salt stress tolerance. Freshly harvested leaves from AhMYB94 transgenic groundnut and WT mock plants were made into discs and immediately immersed in salt solution (1%) for three days in the darkroom. Leaf discs from transgenic groundnut plants showed lower salinity-induced chlorophyll reduction than wild and mock plants. The transgenic plants stayed green, delayed leaf senescence, and displayed better tolerance than the WT and mock plants to salt stress conditions (Fig. 8a).
Salt stress analysis
AhMYB94 transgenic lines showed significant functional leaf area, a lesser extent of chlorosis, salt-induced damage, and delayed leaf senescence. In contrast, wild-type and mock plants show severe necrosis, salt burn leaf edges, and chlorosis (Fig. 8b).