Copalyl Diphosphate Synthase Mutation Improved Salt Tolerance in Maize ( Zea mays. L) via Enhancing Vacuolar Na ⁺ Sequestration and Maintaining ROS Homeostasis

Salinity stress impairs plant growth and causes crops to yield losses worldwide. Reduction of in vivo gibberellin acid (GA) level is known to repress plant size but is beneficial to plant salt tolerance. However, the mechanisms of in vivo GA deficiency-enhanced salt tolerance in maize are still ambiguous. In this study, we generated two independent maize knockout mutant lines of ent-copalyl diphosphate synthase (one of the key enzymes for early steps of GA biosynthesis), zmcps-1 and zmcps-7, to explore the role of GA in maize salt tolerance. The typical dwarf phenotype with lower GA content and delayed leaf senescence under salinity was observed in the mutant plants. The leaf water potential and cell turgor potential were significantly higher in zmcps-1 and zmcps-7 than in the wild type (WT) under salt stress. The mutant plants exhibited a lower superoxide anion production rate in leaves and also a downregulated relative expression level of NAPDH oxidase ZmRbohA-C than the WT maize under salt stress. Also, the mutant plants had higher enzymatic activities of superoxide dismutase (SOD) and catalase (CAT) and higher content of soluble sugars and proline under salt stress. The Na⁺/K⁺ ratio was not significantly different between the mutant maize plants and WT plants under salt stress conditions, but the Na⁺ and K⁺ content was increased in zmcps-1 and zmcps-7 leaves and shoots. Na⁺ fluorescent dye staining showed that the mutant leaves have significantly higher vacuolar Na⁺ intensity than the WT maize. The expression level of vacuolar Na⁺/H⁺ exchanger gene ZmNHX1 and vacuolar proton pump genes ZmVP1-1 and ZmVP2 were upregulated in the zmcps-1 and zmcps-7 plants under salinity, further proving that in vivo GA deficiency enhanced vacuolar Na⁺ sequestration in zmcps-1 and zmcps-7 leaves cells to avoid Na⁺ cytotoxicity. Together, our results suggested that maintaining ROS homeostasis and enhancing vacuolar Na⁺ sequestration could be involved in GA deficiency-improved maize salt tolerance.

Keywords: gibberellin; maize; osmotic adjustment; reactive oxygen species; salt stress; vacuolar sodium sequestration; water potential.