An efficient visualized identification system for characterizing alkaline response genes in soybean

Alkaline stress is one of the most widespread and devastating abiotic stresses and poses a major threat to soybean production. The multi-omics data of soybean have provided abundant genetic information that could be investigated for alkaline stress tolerance improvement. However, the identified alkaline response genes are limited, and gene functional characterization in soybean is dependent on a tissue-culture-based strategy, which is characterized by low efficiency, technical complexity, and time-consuming procedures. Here, we present a simple and efficient system to identify alkaline response genes in a visual manner in soybean. We developed a visualized system for identifying alkaline response genes. A synthetic RUBY cassette, which encodes enzymes converting tyrosine to clearly visible red betalain, was used to induce soybean hairy roots to enable visual selection of transformed tissues. Using this transient identification system, we evaluated a total of 20 differentially expressed genes (DEGs) from previous data. Three alkaline-tolerant genes and two alkaline-sensitive genes were identified based on root growth phenotypes under alkaline stress. On this basis, we further established a multi-gene pyramiding system by expressing three alkaline stress-tolerant genes in a single transcript unit and evaluated the pyramiding effect. The pyramiding lines possessed enhanced alkaline stress tolerance compared with the wild type and single-gene overexpression lines. The visualized identification system is a simple and efficient method enabling the screening of alkaline stress response genes rapidly and accurately. This system can also be applied for preliminary evaluation of candidate genes before generating stable transgenic plants, thereby accelerating functional studies of stress-related genes.