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Literature Sharing | TaSnRK3.23B, a CBL-interacting protein kinase of wheat, confers drought stress tolerance by promoting ROS scavenging in Arabidopsis
Release time:
2025-02-27
Sucrose non-fermenting-1-related protein kinases (SnRKs) have been implicated in plant growth and stress responses. Although SnRK3.23 is known to be involved in drought stress, the underlying mechanism of resistance differs between Arabidopsis and rice, and little is known about its function in wheat.
In the current work, TaSnRK3.23B was detected on the cell membrane and in the nucleus. TaSnRK3.23B overexpression in Arabidopsis promoted reactive oxygen species (ROS) scavenging via the accumulation of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) and then conferred significant tolerance to drought. The prediction analysis, yeast two-hybrid, and bimolecular fluorescence complementation (BiFC) assays revealed that TaSnRK3.23B interacted with TaCBL2B and TaCBL6B, which are calcineurin B-like (CBL) proteins. The predicted model demonstrated that TaSnRK3 subfamily proteins participate in Ca2+ signaling mediated by TaCBL2B/TaCBL6B and subsequently provide drought stress tolerance by promoting ROS scavenging in wheat.
The NJ phylogenetic tree was constructed using BLAST results (Fig. 1A). Detailed information on these proteins is provided in Table S2. The dendrogram demonstrated that the SnRK3 subfamily members were further divided into three subgroups. Three TaSnRK3.23 homologous proteins displayed high homology with CIPK23 of Aegilops tauschii and Triticumurartu. The 20 most statistically significant motifs, named motifs 1 to 20, were selected to depict the motif pattern for the 13 members. The motif analysis revealed that all the 13 members shared certain conserved motifs such as motif1, motif2, motif3, motif4, motif5, motif7, motif8, motif10, and motif11 (Fig. 1B). Specific information about each motif is provided in Table S3. Multiple sequence alignment revealed that 13 members shared conserved domains (Fig. 1C). Homologous TaSnRK3.23 proteins included an identical conserved functional region, demonstrating that the TaSnRK3.23s were evolutionarily conserved and have similar function with CIPK genes in other species.
Fig. 1 Phylogenetic analysis, motif analysis and multiple sequence alignment of TaSnRK3.23s and CIPK23 proteins from different species
TaSnRK3.23B was highly expressed in the two homozygous lines, as confirmed by semi-quantitative PCR (Fig. 3A). Next, we transplanted 4-day-old transgenic and wild-type (WT) seedlings to the 1/2 MS medium supplemented with 0 and 200 mM mannitol, respectively (Fig. 3B). After 7 days, the transgenic seedlings exhibited higher fresh weight and longer root length than the WT seedlings following drought treatment. Compared with the control, no differences were present in fresh weight and root growth between transgenic and WT plants (Fig. 3C and D). Thus, transgenic lines promoted growth following mannitol stress.
Fig. 3 Ectopic expression of TaSnRK3.23B in Arabidopsis afects root growth under mannitol stress
To further confirm whether TaSnRK3.23B was related to drought stress resistance, 4-week-old WT and overexpressing TaSnRK3.23B lines (TaSnRK3.23B-OEs) were subjected to drought stress for 15 days (Fig. 4). No significant difference was recorded in WT and TaSnRK3.23BOEs based on normal conditions (Fig. 4A). However, both WT and TaSnRK3.23B-OEs leaves displayed wilt following drought treatment and WT was more severe than TaSnRK3.23B-OEs and even close to death (Fig. 4B). After rewatering, TaSnRK3.23B-OEs fully restored normal growth, whereas WT completely dried up (Fig. 4C). The results of fresh weight and survival rate demonstrated that transgenic lines had a greater growth than WT under drought treatment. WT lost water more rapidly than the transgenic lines, and thus wilted faster (Fig. 4D,Fig. S7). The growth phenotype data under drought conditions are demonstrated in Table S5. Because drought stress induces membrane lipid peroxidation resulting in membrane system damage, we assessed the content of hydrogen peroxide (H2O2) and malondialdehyde (MDA), and the activity of SOD, POD, and CAT in plants. The contents of MDA and H2O2 of TaSnRK3.23B-OEs significantly decreased compared to WT following drought stress. The activity of SOD, POD, and CAT increased compared to WT under drought stress (Fig. 5, Table S6). The results shown that the cell membrane of WT was more damaged than that of TaSnRK3.23B-OEs, and SOD, POD, and CAT exerted an important regulatory effect on drought stress in transgenic lines.
Fig. 4 Ectopic expression of TaSnRK3.23B positively regulated drought stress tolerance in transgenic Arabidopsis
Subcellular localization experiments demonstrated that N. benthamiana leaf cells containing pCAMBIA2300-TaSnRK3.23B-GFP displayed a high green fluorescence signal in the plasma membrane and nucleus (Fig. 6), suggesting it functions in these regions.
Fig. 6 Subcellular localization of TaSnRK3.23B protein
Protein interaction network predictions revealed that TaSnRK3.23B interacted with TaCBLs (Fig. 7A) and the kinship between them were shown in Table S7. We randomly selected TaCBL2B and TaCBL6B which were closely related with TaCBL3 and TaCBL9 and TaCBL Unknown1 which was closely related with TaCBL1 to examine the interactions with TaSnRK3.23B (Fig. 7B).
Self-activation assay revealed that yeast cells containing BD-TaSnRK3.23B/AD-EV, BD-TaCBL2B/AD-EV, and BD-CBL6B/AD-EV grew on SD-Trp-Leu but not on SD-Trp-Ade-His-Leu, suggesting that TaSnRK3.23B, TaCBL2B, and TaCBL6B exerted no self-activation effect in transgenic yeast cells. Yeast two-hybrid assay confirmed the growth of yeast cells containing BD-TaSnRK3.23B/AD-TaCBL2B and BD-TaSnRK3.23B/AD-TaCBL6B on SD-Trp-Ade-His-Leu plate, suggesting that TaSnRK3.23B interacted with TaCBL2B and TaCBL6B in vitro (Fig. 7C). BiFC assays were conducted to investigate the interaction of the proteins in vivo and the results revealed that TaSnRK3.23B interacted with TaCBL2B and TaCBL6B in the nucleus and cell membrane (Fig. 7D). However, TaCBL-Unknown1 had no self-activation effect in transgenic yeast cells and TaSnRK3.23B could not interacted with TaCBL-Unknown1(Fig. S8).
Fig. 7 TaSnRK3.23B interacts with TaCBL2B and TaCBL6B
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