Dioscorea composita WRKY5 positively regulates AtSOD1 and AtABF2 to enhance drought and salt tolerances.

KEY MESSAGE: DcWRKY5 increases the antioxidant enzyme activity and proline accumulation, oppositely, reduces the accumulation of ROS and MDA, through directly activating the genes expression, finally enhances the salt and drought tolerance. Drought and salinity are two main environmental factors that limit the large-scale cultivation of the medicinal plant Dioscorea composita (D. composita). WRKY transcription factors (TFs) play vital roles in regulating drought and salt tolerance in plants. Nevertheless, the molecular mechanism of WRKY TF mediates drought and salt resistance of D. composita remains largely unknown. Here, we isolated and characterized a WRKY TF from D. composita, namely DcWRKY5, which was localized to the nucleus and bound to the W-box cis-acting elements. Expression pattern analysis showed that it was highly expressed in root and significantly up-regulated in the presence of salt, polyethylene glycol-6000 (PEG-6000) and abscisic acid (ABA). Heterologous expression of DcWRKY5 increased salt and drought tolerance in Arabidopsis, but was insensitive to ABA. In addition, compared with the wild type, the DcWRKY5 overexpressing transgenic lines had more proline, higher antioxidant enzyme (POD, SOD, and CAT) activities, less reactive oxygen species (ROS) and malondialdehyde (MDA). Correspondingly, the overexpression of DcWRKY5 modulated the expression of genes related to salt and drought stresses, such as AtSS1, AtP5CS1, AtCAT, AtSOD1, AtRD22, and AtABF2. Dual luciferase assay and Y1H were further confirmed that DcWRKY5 activate the promoter of AtSOD1 and AtABF2 through directly binding to the enrichment region of the W-box cis-acting elements. These results suggest that DcWRKY5 is a positive regulator of the drought and salt tolerance in D. composita and has potential applications in transgenic breeding.

Dioscorea composita WRKY12 is involved in the regulation of salt tolerance by directly activating the promoter of AtRCI2A.

Dioscorea composita (D. composita) is an important medicinal plant worldwide with high economic value. However, its large-scale cultivation was limited by soil salinization. Identification of genes and their mechanisms of action in response to salt stress are critically important. In the present study, we isolated a classical WRKY transcription factor from D. composita, namely DcWRKY12, and analyzed its function in salt tolerance. Expression pattern analysis showed DcWRKY12 is mainly expressed in roots and significantly induced by NaCl, polyethylene glycol-6000 (PEG-6000), and abscisic acid (ABA). Phenotypic and physiological analyses revealed that heterologous expression of DcWRKY12 enhanced salt and osmotic stress tolerance by increasing antioxidant enzyme activity, osmoregulatory substance content, maintaining relative water content and ion homeostasis, decreasing reactive oxygen species and malondialdehyde content. Correspondingly, the overexpression of DcWRKY12 modulated the expression of salt stress-responsive and ion transport-related genes. Dual luciferase assay and Y1H were further confirmed that DcWRKY12 activates the promoter of AtRCI2A through directly binding to the specific W-box cis-acting elements. These results suggest that DcWRKY12 is a positive regulator of salt tolerance in D. composita and has potential applications in salt stress.

Dioscorea composita WRKY3 positively regulates salt-stress tolerance in transgenic Arabidopsis thaliana.

Dioscorea composita (D. composita) is a perennial dioecious herb with strong biotic and abiotic stress tolerance. However, what roles WRKY transcription factors might play in regulating abiotic stress responses in this medicinal plant is unknown. Here, we isolated DcWRKY3 from D. composita and analyzed its role in stress tolerance. DcWRKY3 is a group I WRKY transcription factor that localized to the nucleus and specifically bound to the W-box cis-elements, but lacked transcriptional activation activity in yeast cells. The expression of DcWRKY3 was strongly affected by salt stress. The heterologous expression of DcWRKY3 strongly enhanced the seed germination rate and root length of Arabidopsis thaliana under salt stress. The DcWRKY3-expressing transgenic lines (DcWRKY3-OEs) also showed higher proline content and antioxidant enzyme activity but lower malondialdehyde and reactive oxygen (ROS) levels compared with the wild type. Moreover, these plants showed upregulated expression of genes related to the salt-stress response and ROS clearance. These findings indicate that DcWRKY3 plays a positive role in the salt-stress response by improving the ROS scavenging ability and maintaining the balance of osmotic pressure in plants. Further studies showed that DcWRKY3 binds to the promoter of AtP5CS1, but not AtSOD and AtRD22, suggesting that DcWRKY3 improves salt tolerance in plants by directly or indirectly regulating the expression of downstream genes. This functional characterization of DcWRKY3 provides new insight into the molecular mechanism underlying the response of D. composita to salt stress.