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 GmHdz4 was isolated. Chimeric soybean plants, GmHdz4 overexpressing (GmHdz4-oe), and gene-editing via CRISPR/Cas9 (gmhdz4) in hairy roots, were generated to examine the GmHdz4 gene response to polyethylene glycol (PEG)-simulated drought stress. Bioinformatic analysis showed GmHdz4 belonged to clade δ, and was closely related to other drought tolerance-related HD-Zip I family genes such as AtHB12, Oshox12, and Gshdz4. The GmHdz4 was located in the plant nucleus and showed transcriptional activation activity by yeast hybrid assay. Quantitative real-time PCR analysis revealed that GmHdz4 expression varied in tissues and was induced by PEG-simulated drought stress. The gmhdz4 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 GmHdz4-oe 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 gmhdz4 compared with both GmHdz4-oe and NT. Together, our results provide new insights for future research on the mechanisms by which GmHdz4 gene-editing via CRISPR/Cas9 system could promote drought stress and provide a potential target for molecular breeding in soybean.

Keywords: Glycine max; GmHdz4; HD-ZIP; drought stress; root system architecture.

Figure 1

Sequence alignment and phylogenetic analysis. (A) Multiple sequence alignment for GmHdz4 and other drought stress-related HD-Zip I proteins. HD and LZ domains are underlined by black and gray, respectively. The corresponding IDs of Oshox22, Oshox4, AtHB7, AtHB12, AtHB6, Gshdz4, GmHdz4, HAHB4, Zmhdz10, SlHZ48, and SlHZ5, are Q7XUJ5.2, Q6K498.1, P46897.2, Q9M276.1, P46668.1, AOG74801, Glyma11g06940, XP_022022563, AFT92045, XP_004245456, and XP_004230017, respectively. (B) Phylogenetic analysis of HD-Zip I transcription factors from Arabidopsis, rice, maize, tomato, soybean, and wild soybean. The phylogenetic tree was built using NJ method in MEGA. Bootstrap support is indicated on the branches. Genes from each of the species are marked with different symbols: Arabidopsis (○), rice (■), maize (□), soybean (▼), wild soybean (▽), and tomato (●).

Figure 2

Tissue-specific and spatio-temporal expression of GmHdz4. Expression pattern of GmHdz4 in various tissues in soybean (A) and PEG-induced expression pattern of GmHdz4 in root (B). Significant differences are indicated by ^* and ^** for p < 0.05 and p < 0.01, respectively, according to Duncan’s test in panel A; values with different letters (a–d) significantly differ at p < 0.05 according to Duncan’s test in panel B.

Figure 3

Subcellular localization analysis of GmHdz4. The fusion plasmid (35S::GmHdz4–GFP), the negative control plasmid (35S::mGFP), and pBWD-NLSmKAT expressing an NLS Red Marker protein were transiently transformed into tobacco epidermal cells. Images were taken in the dark field for green fluorescence (a1, b1), red fluorescence (a2, b2), and chloroplast fluorescence (a3, b3), while the outline of cells (a4, b4) and merged image (a5, b5) were photographed in a bright field. Microscopy detections of 35S::mGFP were displayed in penal (A), and the detections of 35S::GmHdz4–mGFP were displayed in penal (B). Bars represent 20 μm.

Figure 4

Transcription activity analysis of GmHdz4. (A) The construct of pGBKT7–GmHdz4; (B) ADE2 and HIS3 reporter assay, and galactosidase (LacZ) assay. pGADT7-T/pGBKT7-53 and pGADT7-T/pGBKT7-Lam vector groups were used as positive and negative controls, respectively.

Figure 5

The phenotype and root system architecture among NT, gmhdz4 chimeric lines, and GmHdz4-oe chimeric lines. Phenotypic comparison of transgenic hairy roots chimera and NT soybeans before and after PEG treatment (A1–F2). The root system architecture analysis of total root length (G), superficial area (H), average diameter (I), and tip numbers (J). NT, non-transgenic soybeans; gmhdz4, gene-editing chimeric line; GmHdz4-oe, overexpression GmHdz4 chimeric line. At least three biological replicates were performed, and the data before (0d) and after (8 days) treatment were statistically analyzed, respectively. Significant differences are indicated by ^* and ^** for p < 0.05 and p < 0.01, respectively, according to Duncan’s test.

Figure 6

The dry matter mass (A) and root–shoot ratio (B) of each line before and after PEG treatment. Significant differences are indicated by ^* for p < 0.05, according to Duncan’s test.

Figure 7

Soluble sugar content (A), proline content (B), and root activity (C) after PEG treatment. The straight line, dotted line, and dash-dotted line represent non-transgenic soybeans (NT), gmhdz4 chimeric line, and GmHdz4-oe line, respectively.

Figure 8

Knockout of GmHdz4 promotes ROS scavenging in response to drought stress. Contents of H₂O₂ (A), O²⁻ (B), and malondialdehyde (MDA) (C), and activities of catalase (CAT) (D), superoxide dismutase (SOD) (E), and peroxidase (POD) (F) were tested to assess the antioxidation systems under drought stress. Significant differences are indicated by ^* and ^** for p < 0.05 and p < 0.01, respectively, according to Duncan’s test.