The bZIP transcription factor TabZIP15 improves salt stress tolerance in wheat

As one of the most important staple foods, wheat plays a crucial role in sustaining food security. However, its production is greatly threatened by abiotic stresses such as salt (Khataar et al., 2018; Qi et al., 2019). Therefore, discovering the genes involved in abiotic stress tolerance and cultivating genetically modified plant varieties with enhanced stress tolerance are currently among the most important goals for plant breeders (Genc et al., 2019). To identify wheat transcriptional factor (TF) genes that are essential for regulating abiotic stress tolerance, 1455 wheat TF-encoding genes from full-length cDNA sequence libraries were tested for their expression under abiotic stress treatment conditions. One gene, TabZIP15 (TraesCS7A02G488600), was identified as a salt stress-responsive gene in wheat. TabZIP15 contains six exons and five introns in its structure (Figure 1a) and encodes a bZIP TF (Figure 1b). In the roots at the seedling stage, TabZIP15 expression was induced quickly and peaked within 1 h following salt stress treatment, and the expression level was approximately 2.4-fold greater than that before treatment (Figure 1c). TabZIP15 is a nuclear-localized protein (Figure 1d) with transcriptional activation activity in yeast (Figure 1e). Moreover, we also found that TabZIP15 has an affinity for the ABA-responsive element (ABRE) Cis-element using the yeast one-hybrid assay system (Figure 1f). To determine the role of the TabZIP15 gene in the regulation of salt stress tolerance in wheat, transgenic lines of the wheat variety Kenong 199 (KN199) overexpressing TabZIP15 under the control of the maize Ubi promoter were generated. Under normal conditions, no obvious phenotypic variation was detected between KN199 plants and the transgenic lines. However, after 20 days of salt stress treatment with 300 mm NaCl, although both the KN199 and transgenic plants displayed a certain degree of wilting and inhibited growth, the wilting degree was obviously lower in the transgenic plants than in the KN199 plants (Figure 1g). In addition, the transgenic plants presented obvious increases in height and aboveground fresh weight, and significant decreases in malondialdehyde (MDA) and H2O2 contents (Figure 1h-k). Considering that the root system is closely related to plant abiotic stress tolerance, we also examined the morphology of the roots and found no obvious differences in root morphology, length or fresh weight between the KN199 plants and transgenic plants under normal growth conditions. However, under salt stress conditions, although the root growth of both the transgenic plants and KN199 plants was inhibited, the transgenic plants had a larger root system than the KN199 plants (Figure 1l), and the root length and fresh weight were greater in the transgenic lines than in the KN199 plants (Figure 1m and n). In the reproductive stage, the spikes of the transgenic plants were almost the same as those of the KN199 plants under normal growth conditions, while the spike length was longer and the number of grains per spike was significantly higher in the transgenic plants than in the KN199 plants (Figure 1o-q). Together, these results suggest that the TabZIP15 gene is involved in the regulation of wheat salt stress tolerance. To better understand how TabZIP15 is involved in the wheat stress response, the truncated TabZIP15 was used to screen the yeast library. The results showed that the enolase protein TaENO-b (KC342470.1) could physically interact with TabZIP15 (Figure 1r). We then conducted luciferase complementation imaging and bimolecular fluorescence complementation assays in Nicotiana benthamiana and confirmed the interaction between TabZIP15 and TaENO-b (Figure 1s and t). TaENO-b is an enolase protein that catalyses the reversible dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate as part of the glycolytic and gluconeogenesis pathways. Together, these results indicated that TabZIP15 may interact with enolase TaENO-b to participate in the regulation of the glycolysis and gluconeogenesis pathways, thus improving salt stress tolerance in wheat. To further investigate the putative mechanisms of the TabZIP15 gene, we used RNA sequencing (RNA-seq) transcriptomic analysis to identify differentially expressed genes (DEGs) (|log2 fold change [FC]| > 1, false discovery rate [FDR] < 0.05) between the KN199 and H381 transgenic plants before (0 h) and after salt stress treatment (1 h) at the seedling stage (Audic and Claverie, 1997; Mariani et al., 2003). As a result, a total of 3332 DEGs were found between KN199 and H381 plants at 0 h, including 1297 up-regulated and 2035 down-regulated DEGs in H381. At 1 h, 2807 DEGs were identified between KN199 and H381 plants, including 2691 up-regulated and 116 down-regulated DEGs in H381 plants (Figure 1u). We then tested the expression of four genes by qRT-PCR, and the results were consistent with the transcriptome analysis results (Figure 1v). Considering that the genes whose expression was specifically induced or inhibited after salt stress treatment may have more important roles in regulating salt tolerance in the transgenic plants, we further compared the DEGs between 0 h and 1 h. The results showed that the expression of 2556 DEGs was specifically up-regulated after salt treatment, while the expression of 45 DEGs was specifically down-regulated (Figure 1w). The large difference in the numbers of up-regulated and down-regulated genes may reflect TabZIP15’s role as a transcriptional activator. Gene Ontology (GO) enrichment analysis was performed for the DEGs that were specifically up-regulated at 1 h in transgenic plants. The 2556 genes were mainly enriched in 20 biological processes, including metabolic processes (GO:0008152), iron ion binding (GO:0005506) and the response to abiotic stimuli (GO:0009628; Figure 1x). Among these GO terms, the metabolic processes item enriched more than 900 DEGs, suggesting that the genes involved in metabolic regulation may play important roles in improving the salt tolerance of wheat through TabZIP15. Furthermore, more than 30 DEGs were enriched in the response to abiotic stimulus GO term. These DEGs may also regulate salt stress tolerance in wheat. In the future, we will analyse these DEGs in depth, which will help us to further understand the mechanism of the TabZIP15 gene regulating salt tolerance in wheat. Discovering stress-responsive genes and studying their roles via genetic modification approaches is necessary for the development of plants with enhanced stress tolerance (Abhinandan et al., 2018; Hasegawa et al., 2000; Zhu, 2016). In this study, we characterized a salt-induced bZIP gene, TabZIP15, in wheat. Based on the performance of TabZIP15 in transgenic plants, we propose that manipulating the expression of this gene leads to enhanced salt stress tolerance. These results improve our understanding of the roles of wheat bZIP protein in the plant response to abiotic stress and highlight a candidate gene for wheat improvement. We thank Jizeng Jia and Ruilian Jing's groups for providing FLC and yeast library. This work was supported by the National Science Foundation (31771792), the National Transgenic Research Project (2016ZX08009001-001-004), the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences and the Tianshan Innovation Team Plan (2020D14002). The authors declare no conflict of interest. X.K. and L.Z. conceived the study and designed the experiments. C.B., C.D., Y.Y., C.J., Y.Y., Y.Z., C.X. and F.D. performed the experiments. L.Z., X.K., Z.N., C.B., Y.Y. and Y.Y. wrote the manuscript.