McWRKY57-like isolation and sequence analysis
Previous studies have confirmed that WRKY57 positively regulates drought tolerance in Arabidopsis [28, 29]. To identify putative WRKY genes related to drought tolerance in mint, we performed a BLASTP search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) on the reported transcriptome data (SRP132644) at the National Center for Biotechnology Information (NCBI) [33] using the AtWRKY57 amino acid sequence as a query. A WRKY57 homologous gene with an open reading frame of 855 codons and encoding 284 amino acid residues was screened out from M. canadensis L. and named McWRKY57-like (Fig. 1A). Phylogenetic tree analysis of Arabidopsis WRKY family proteins showed that McWRKY57-like clustered with AtWRKY57, which belongs to the IIc subgroup of the WRKY family (Fig. 1B). Sequence alignment of McWRKY57-like with other WRKY57 homologous proteins from Arabidopsis, Glycine max, Solanum lycopersicum, Gossypium raimondii, Fragaria vesca, and Oryza sativa showed that McWRKY57-like contains a highly conserved WRKYGQK domain and a C2H2-type (C-X4–5-C-X22–23-H-X1-H) zinc finger motif (Figs. 1C and D), indicating that McWRKY57-like has a close relationship with the WRKY57 ortholog of Arabidopsis thaliana (Fig. 1D).
Subcellular Localization And Transcriptional Activity Of Mcwrky57-like
To determine the subcellular localization of McWRKY57-like, we constructed the 35S::McWRKY57-like-GFP plasmid, in which McWRKY57-like was fused to the N-terminus of the green fluorescent protein (GFP) gene under the control of the 35S promoter. Then, the recombinant vector was transiently co-expressed in Nicotiana benthamiana leaves with a nuclear marker 35S::D53-RFP [34]. The 35S::McWRKY57-like-GFP construct was further transformed into Arabidopsis wild-type (WT) plants, and the subcellular location of McWRKY57-like in the transgenic seedlings was observed under a confocal microscope after DAPI staining. The observations reveal that McWRKY57-like is expressed in the nucleus (Fig. 2A and B).
We further investigated the transcriptional activation activity of McWRKY57-like using a yeast assay system. The full-length coding sequence of McWRKY57-like was introduced into the pGBKT7 vector to generate the BD-McWRKY57-like, which was then transformed into yeast strain Y2H. The yeast strains containing pGBKT7 (BD vector) and pGBKT7-AtSIZ1 (BD-AtSIZ1) were used as the empty and positive controls, respectively. As shown in Fig. 2C, all yeasts in our experiment grew normally on SD/-Trp medium, but only yeast transformed with the BD-McWRKY57-like or BD-AtSIZ1 could grow normally on SD/-Trp/-Leu/-Ade medium. These results confirm that McWRKY57-like has transcription factor activity.
Analysis of McWRKY57-like gene expression in different tissues
To investigate the potential working locations of McWRKY57-like gene in M. canadensis, we detected McWRKY57-like transcript levels in different tissues, including young leaves, mature leaves, flowers, stems, and roots using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results showed that MhWRKY57-like gene was expressed the most in stems, followed by flowers, roots, young leaves, and mature leaves in descending order (Fig. 3A). To further determine the MhWRKY57-like expression pattern, we constructed a GUS vector driven by the MhWRKY57-like promoter, which was obtained using Genome Walking Kit (Figure S1), and introduced it into WT Arabidopsis plants. One transgenic ProMhWRKY57−like-GUS line was selected to analyze the expression of MhWRKY57-like gene. GUS expression was detectable by histochemical staining in young seedlings, leaves, stems, flowers, young siliques, and mature siliques, but not in mature seeds of the transgenic Arabidopsis (Fig. 3B–H), showing the strongest GUS staining in flowers and stems (Fig. 3B and C). GUS expression was also detectable in rosette leaves, where leaf veins exhibited stronger GUS staining than other parts of leaves (Fig. 3D). Moreover, GUS staining was also intense in veins of cotyledons of young seedlings (Fig. 3E). Unexpectedly, GUS staining was undetectable in the roots of transgenic Arabidopsis seedlings. Interestingly, GUS expression was higher in young siliques (about 3 days after pollination) than in mature siliques (about 10 days after pollination) and was not detectable in mature seeds (Fig. 3F–H). These results suggest that MhWRKY57-like is significantly expressed in flowers, stems, and roots of mint, showing a different pattern from transgenic Arabidopsis plants.
McWRKY57-like expression under different treatments
Genes in the WRKY family play pivotal roles in plant response to abiotic stresses, and their expression levels are influenced by environmental factors and hormones [20]. To further analyze the potential functions of McWRKY57-like gene, we performed qRT-PCR to examine its expression level in leaves and roots after mannitol, NaCl, abscisic acid (ABA), and methyl jasmonate (MeJA) treatments. The results showed that 1) when treated with 300 mM mannitol, McWRKY57-like expression was markedly induced in leaves at 8, 12, and 24 hours after treatment (h.a.t), reaching a peak at 8 h.a.t at about a 33-fold increase, and it was increased in roots by about 8-fold within 2–24 h, reaching a peak at 2 h.a.t (Fig. 4A and B); 2) under treatment with 150 mM NaCl, McWRKY57-like expression was increased in leaves within 2–24 h.a.t, reaching a peak at 24 h.a.t at about a 27-fold increase, and increased in roots by about 2-fold at 2 h.a.t and did not change too much in the subsequent 4–24 h.a.t (Fig. 4C and D); 3) when treated with ABA, McWRKY57-like expression decreased in leaves at 2–12 h.a.t. while increased observably by almost 3 fold at 24 h.a.t., and enhanced remarkably in roots at each time point, reaching a peak by about 4 fold at 24 h.a.t. (Fig. 4E and F); and 4) Under the MeJA treatment, McWRKY57-like expression increased significantly in leaves at 8 and 12 h.a.t. and enhanced notably in roots at 8 and 24 h.a.t. (Figs. 4G and H). Among these four treatments, mannitol treatment induced McWRKY57-like expression to the highest level in both leaves and roots. We also examined McWRKY57-like expression in leaves under natural drought stress and found that its mRNA level was increased by nearly 7-fold compared with the control (Fig. 4I). These results suggest that McWRKY57-like gene responds most intensely to drought stress and may play a role in regulating drought stress in plants.
Overexpression of McWRKY57-like in Arabidopsis increases drought tolerance
To further investigate whether McWRKY57-like contributes to drought resistance, the 35S::McWRKY57-like-GFP construct was transformed into Arabidopsis WT plants. Two homozygous McWRKY57-like overexpression lines, McWRKY57-like OE-8 and OE-10, were obtained, and McWRKY57-like expression was assessed in transgenic seedlings using RT-PCR (Fig. 5A). We next performed drought treatments for plants at seedling stage. We observed no significant difference between WT and McWRKY57-like OE lines grown on 1/2 MS without 300 mM mannitol treatment (Fig. 5B and D). In contrast, we observed longer roots in McWRKY57-like OE lines than in WT plants grown on 1/2 MS with 300 mM mannitol (Fig. 5C and D).
To further investigate the function of McWRKY57-like in plant vegetative growth, WT and transgenic plants grown in soils for 3 weeks were subjected to natural drought stress. After 10 days of natural drought treatment, most leaves maintained green in OE-8 and OE-10 transgenic lines but withered and chlorotic in WT plants (Fig. 5E). We further measured leaf chlorophyll content in WT and McWRKY57-like OE lines. We found no significant difference in leaf chlorophyll content between WT and McWRKY57-like OE lines under control conditions. However, the leaf chlorophyll content was significantly higher in McWRKY57-like OE plants than in WT plants after natural drought treatment (Fig. 5F). Reducing water loss is advantageous in enhancing drought tolerance. Therefore, we analyzed leaf water loss rates in WT and McWRKY57-like OE plants. The results showed that both McWRKY57-like OE lines (OE8 and OE10) exhibited evidently lower water loss rates than WT under natural drought stress (Fig. 5G). These results suggest that McWRKY57-like overexpression enhances drought tolerance of transgenic Arabidopsis.
McWRKY57-like overexpression in Arabidopsis improves osmolyte accumulation and antioxidant enzyme activities
Drought stress can cause osmotic stress in plants. Under osmotic stress, plants can synthesize some osmotic-regulating substances. To further elucidate the mechanism by which McWRKY57-like enhances drought tolerance, the contents of proline, soluble sugar, and soluble protein were detected in WT and McWRKY57-like OE plants under normal and drought conditions. We found that the contents of proline, soluble sugar, and soluble protein were not evidently different between WT and McWRKY57-like OE plants under control conditions but were significantly increased in both WT and McWRKY57-like OE plants under drought stress. Furthermore, these increases were more pronounced in McWRKY57-like OE lines (Fig. 6A–C). These results indicate that McWRKY57-like overexpression enhances the osmoregulation capacity of transgenic plants under drought stress.
Drought stress can also induce the accumulation of reactive oxygen species (ROS), such as superoxide anion radicals (O2−) and hydrogen peroxide (H2O2), leading to oxidative damage to plant cells [35]. Malondialdehyde (MDA) is a product of ROS-stimulated lipid peroxidation, and MDA content is commonly used to assess the extent of ROS-mediated damage to plants [36]. We examined MDA content in plants under control and drought conditions. The results showed that MDA content increased in WT and McWRKY57-like OE plants after drought treatment, and the increase was remarkably higher in WT than in McWRKY57-like OE lines (Fig. 6D). We also tested the enzymic activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), the key enzymes for scavenging ROS. The results showed that their activities were evidently increased under drought stress in McWRKY57-like OE plants than in WT plants (Fig. 6E–G). These results suggest that McWRKY57-like overexpression enhances the activities of antioxidant enzymes in transgenic plants under drought stress.
McWRKY57-like overexpression upregulates the expression of the stress-responsive genes
To investigate the mechanisms by which McWRKY57-like overexpression enhances drought resistance in Arabidopsis, we further performed qRT-PCR to analyze the expression levels of some stress-responsive genes in transgenic Arabidopsis, including AtRD29A, AtRD29B, AtRD20, AtRAB18, AtCOR15A, AtCOR15B, AtKIN2, and AtDREB1A. The expression levels of AtRD29A and AtRD29B were not significantly different between McWRKY57-like OE lines and WT plants under normal growth condition but were upregulated in both OE lines and WT plants under drought treatment. However, the increase was more pronounced in McWRKY57-like OE lines than in WT plants (Fig. 7A and B). Furthermore, the transcript levels of AtRD20, AtRAB18, AtCOR15A, AtCOR15B, AtKIN2, and AtDREB1A were significantly downregulated in McWRKY57-like OE lines compared to WT plants under control conditions. When treated with drought stress, the transcript levels of these genes were evidently upregulated in McWRKY57-like OE and WT plants, with markedly higher levels in McWRKY57-like OE lines than in WT plants (Fig. 7C–H). The upregulation of these drought-related genes may partly explain the enhanced tolerance of McWRKY57-like OE transgenic plants.