Soybean (Glycine max (L.) Merr.), is one of the world’s most important oilseed and economic crops, yet its cell-biology-oriented gene-function studies remain hampered by low transformation efficiency and poor detection of fluorescent tags such as GFP. Researchers therefore routinely resort to heterologous systems like Arabidopsis or Nicotiana benthamiana, risking mis-localization and artifactual activity of soybean proteins and compromising physiological relevance. A robust, soybean-based platform is urgently needed. In this study, we introduce FLASH (Fluorescent Localization Assay for Subcellular in Hairy-root-derived callus protoplasts). Transgenic hairy roots expressing fluorescent proteins are bulked into callus, from which protoplasts are isolated in a single step and > 80% of cells display bright, stable fluorescence within 24 h. Using FLASH we monitored, in real time, light-induced assembly of GmCRY photobodies in both the nucleus and cytoplasm, and confirmed nuclear co-localization of GmCRY1b with STF1. FLASH offers a high-fidelity, high-throughput solution for subcellular localization, protein–protein interaction, and phase-separation studies of soybean proteins.

WUSCHEL-related homeobox (WOX) transcription factors play a crucial role in lateral organ development in several plant species; however, their precise functions in soybean (Glycine max [L.] Merr.) were unclear. Here, we identified two independent multi-leaflet soybean mutants, mlw48-8 and mlw48-161, from a CRISPR/Cas9-engineered mutant library in the Williams 82 background. Both mutants exhibited irregular leaf margins, and the upper leaves were narrow and almost lanceolate at maturity. Molecular analysis revealed that these are allelic mutants with independent mutations in the WUSCHEL-related homeobox1 (GmWOX1A) gene. A transcriptome analysis demonstrated that GmWOX1A modulates the expression of auxin- and leaf development–related genes. Yeast two-hybrid and split-luciferase complementation imaging assays revealed that GmWOX1A interacts with the YABBY family protein GmYAB5, providing further evidence of its potential involvement in leaf development. Notably, the mlw48-161 mutant showed an increased seed number per plant. Consequently, our study not only provides valuable insights into the role of GmWOX1A in soybean leaf development but also offers a potential strategy for high-yield breeding.

Soybean (Glycine max) responds to ambient light variation by undergoing multiform morphological alterations, influencing its yield potential and stability in the field. Phytochromes (PHYs) are plant-specific red (R) and far-red (FR) light photoreceptors mediating photomorphogenesis and photoperiodic flowering. As an ancient tetraploid, soybean harbors four PHYA, two PHYB, and two PHYE paralogs. Except for GmPHYA2/E4 and GmPHYA3/E3, which have been identified as photoperiod-dependent flowering repressors, the functions of GmPHYs are still largely unclear. We generated a series of individual or combined mutations targeting the GmPHYA or GmPHYB genes using CRISPR/Cas9 technology. Phenotypic analysis revealed that GmPHYB1 mediates predominantly R-light induced photomorphogenesis, whereas GmPHYA2/E4 and GmPHYA3/E3, followed by GmPHYA1 and GmPHYB2, function redundantly and additively in mediating FR light responses in seedling stage. GmPHYA2/E4 and GmPHYA3/E3, with weak influence from GmPHYA1 and GmPHYA4, delay flowering time under natural long-day conditions. This study has demonstrated the diversified functions of GmPHYAs and GmPHYBs in regulating light response, and provides a core set of phytochrome mutant alleles for characterization of their functional mechanisms in regulating agronomic traits of soybean.