CRISPR/Cas9 mediated gene-editing of GmHdz4 transcription factor enhances drought tolerance in soybean (Glycine max [L.] Merr.)

Abstract

The HD-Zip transcription factors play a crucial role in plant development, secondary metabolism, and abiotic stress responses, but little is known about HD-Zip I genes in soybean. Here, a homeodomain-leucine zipper gene designated <i>GmHdz4</i> was isolated. Chimeric soybean plants, <i>GmHdz4</i> overexpressing (<i>GmHdz4-oe</i>), and gene-editing <i>via</i> CRISPR/Cas9 (<i>gmhdz4</i>) in hairy roots, were generated to examine the <i>GmHdz4</i> gene response to polyethylene glycol (PEG)-simulated drought stress. Bioinformatic analysis showed <i>GmHdz4</i> belonged to clade δ, and was closely related to other drought tolerance-related HD-Zip I family genes such as <i>AtHB12</i>, <i>Oshox12</i>, and <i>Gshdz4</i>. The <i>GmHdz4</i> was located in the plant nucleus and showed transcriptional activation activity by yeast hybrid assay. Quantitative real-time PCR analysis revealed that <i>GmHdz4</i> expression varied in tissues and was induced by PEG-simulated drought stress. The <i>gmhdz4</i> showed promoted growth of aboveground parts, and its root system architecture, including the total root length, the root superficial area, and the number of root tips were significantly higher than those of <i>GmHdz4-oe</i> even the non-transgenic line (NT) on root tips number. The better maintenance of turgor pressure by osmolyte accumulation, and the higher activity of antioxidant enzymes to scavenge reactive oxygen species, ultimately suppressed the accumulation of hydrogen peroxide (H₂O₂), superoxide anion (O^2-), and malondialdehyde (MDA), conferring higher drought tolerance in <i>gmhdz4</i> compared with both <i>GmHdz4-oe</i> and NT. Together, our results provide new insights for future research on the mechanisms by which <i>GmHdz4</i> gene-editing <i>via</i> CRISPR/Cas9 system could promote drought stress and provide a potential target for molecular breeding in soybean.