PpCML14 from Kentucky bluegrass confers drought and salinity tolerance in transgenic rice

PpCML14 confers drought and salinity tolerance by interacting with PpGMP1 for improved AsA biosynthesis and upregulating antioxidant enzyme activities, proline accumulation, and stress-responsive genes. Calcium is a universal second messenger in plant cells, regulating plant growth, development, and responses to environmental stresses. Calmodulin-like proteins (CMLs) are one of the Ca²⁺ sensors or Ca²⁺-binding proteins. However, the functions of lots of members in CML family remain largely unknown. A PpCML14 from the native Kentucky bluegrass (Poa pratensis L.) was examined to regulate drought and salinity tolerance in the present study. PpCML14 is highly expressed in roots, and its overexpression in rice resulted in increased drought and salinity tolerance, with promoted activities of antioxidant enzymes, including superoxide dismutase, catalase, and ascorbate peroxidase, proline accumulation, and expressions of ABA-dependent and ABA-independent stress-responsive genes. Additionally, PpGMP1 (GDP-D-mannose pyrophosphorylase 1), a key enzyme for ascorbic acid (AsA) biosynthesis, was identified as interacting with PpCML14 based on screening of cDNA library and further confirmation using the methods of yeast-two-hybridization, firefly luciferase complementation imaging (LCI), pull-down, and co-immunoprecipitation (Co-IP). Overexpression of PpGMP1 in rice led to increased drought and salinity tolerance. AsA levels and AsA redox were higher, but reactive oxygen species (ROS) accumulation was lower in both PpGMP1- and PpCML14-overexpressing rice lines under drought and salinity conditions compared with wild-type plants. The results indicated that AsA biosynthesis is regulated by the PpCML14-PpGMP1 module. AsA is an important antioxidant for scavenging ROS. It is suggested that PpCML14 confers drought and salinity tolerance through upregulating antioxidant enzyme activities, proline accumulation, and stress--responsive genes, and by activating PpGMP1 to improve AsA biosynthesis.