SbNAC9 Improves Drought Tolerance by Enhancing Scavenging Ability of Reactive Oxygen Species and Activating Stress-Responsive Genes of Sorghum

Abstract

Drought stress severely threatens the yield of cereal crops. Therefore, understanding the molecular mechanism of drought stress response of plants is crucial for developing drought-tolerant cultivars. NAC transcription factors (TFs) play important roles in abiotic stress of plants, but the functions of NAC TFs in sorghum are largely unknown. Here, we characterized a sorghum NAC gene, SbNAC9, and found that SbNAC9 can be highly induced by polyethylene glycol (PEG)-simulated dehydration treatments. We therefore investigated the function of SbNAC9 in drought stress response. Sorghum seedlings overexpressing SbNAC9 showed enhanced drought-stress tolerance with higher chlorophyll content and photochemical efficiency of PSII, stronger root systems, and higher reactive oxygen species (ROS) scavenging capability than wild-type. In contrast, sorghum seedlings with silenced SbNAC9 by virus-induced gene silencing (VIGS) showed weakened drought stress tolerance. Furthermore, SbNAC9 can directly activate a putative peroxidase gene SbC5YQ75 and a putative ABA biosynthesis gene SbNCED3. Silencing SbC5YQ75 and SbNCED3 led to compromised drought tolerance and reduced ABA content of sorghum seedlings, respectively. Therefore, our findings revealed the important role of SbNAC9 in response to drought stress in sorghum and may shed light on genetic improvement of other crop species under drought-stress conditions.

Keywords: NAC transcription factor; Sorghum bicolor; drought stress; reactive oxygen species (ROS); virus induced-gene silencing.

Figure 1

SbNAC9 responds to PEG-simulated dehydration stress. (A) Relative expression of SbNAC9 in the fourth leaf of sorghum seedlings at four-leaf stage under hormone and abiotic stress treatments. Sorghum seedlings were used for water (Mock), 150 μM GA (gibberellin), 150 μM ABA, 200 mM NaCl, 200 mM mannitol, and 20% PEG6000 treatments. For low temperature treatment, sorghum seedlings were kept at 4 °C. SbEIF4A was used as the internal reference. Error bars indicate SD of three independent experiments. ** p < 0.01 and * p < 0.05 by Student’s t-test. (B) Time course transcript level of SbNAC9 in sorghum seedlings at four-leaf stage subjected to 20% PEG treatment. SbEIF4A was used as the internal reference. Error bars indicate SD of three independent experiments. (C) Relative transcript level of SbNAC9 in various tissues of sorghum at filling stage. SbEIF4A was used as the internal reference. Error bars indicate SD of three independent experiments. (D) Relative expression level of SbNAC9 in the three-day-old root tips of sorghum under MS media soaked into 20% PEG solution for 4 h and 6 h. SbEIF4A was used as the internal reference. Error bars indicate SD of three independent experiments. ** p < 0.01 by Student’s t-test. (E) In situ hybridization assay of SbNAC9 in root tips of the three-day-old sorghum seedlings subjected to 20 % PEG-simulated drought-stress treatment for 4 h and 6 h. Root tips grown on MS media served as negative control. Bars indicate 100 μm.

Figure 2

SbNAC9 functions as a transcriptional activator. (A) Schematic diagrams of constructs in SbNAC9 transcriptional activation assay. (B) Transcriptional activation of SbNAC9. Yeast cells were diluted to 1, 10⁻¹, 10⁻² from the left panel to the right panel. pBD-SbNAC9-FL, full-length CDS of SbNAC9; pBD-SbNAC9-N, N-terminal of SbNAC9; pBD-SbNAC9-C, C-terminal of SbNAC9; and pBD, pGBKT7-BD vector. pGBKT7-BD was used as a negative control. (C) Subcellular localization of SbNAC9 in tobacco leaves. DAPI (2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride) channel represents the signal of nucleus colored in blue. GFP channel represents the signal of SbNAC9-GFP or GFP colored in green. Merge indicates combination of GFP (green fluorescent protein), DAPI, and Bright together colored in cyan. Bars indicate 50 μm.

Figure 3

Overexpression of SbNAC9 enhanced drought-stress tolerance of sorghum. (A) Phenotype of sorghum plants under drought-stress treatment. Three-week-old WT and transgenic lines (OE-1, OE-9, OE-14) were treated with water deprivation for 21 days. Bars indicate 10 cm. (B) Phenotype of first five leaves of WT and transgenic lines after 21-day drought-stress treatment. Bars indicate 5 cm. (C,D) Chlorophyll content (C) and chlorophyll fluorescence Fv/Fm (D) in the fifth leaf of WT and transgenic lines after drought treatment. Error bars indicate SD of three independent experiments. * p < 0.05 by Student’s t-test. (E) DAB and NBT staining of leaves of WT and transgenic sorghum seedlings treated with drought stress for 5 days. Bars indicate 5 cm. (F–H) POD activity (F), SOD activity (G), and MDA content (H) of WT and transgenic lines after drought-stress treatment. Error bars indicate SD of three independent experiments. * p < 0.05 by Student’s t-test.

Figure 4

Silenced SbNAC9 weakened drought-stress tolerance of sorghum. (A) Phenotype of sorghum seedlings inoculated with BSMV:00 and BSMV:SbNAC9 under mock and drought-stress treatments. Bars indicate 6 cm for the whole plants in the left panels and 2 cm for the leaves in the middle and right panels. (B) Relative transcript level of SbNAC9 in sorghum seedlings silenced by VIGS. SbEIF4A was used as the internal reference. Error bars indicate SD of three independent experiments. **** p < 0.0001 and *** p < 0.001 by Student’s t-test. (C,D) Chlorophyll content (C) and chlorophyll fluorescence Fv/Fm (D) in the third leaf of sorghum seedlings inoculated with BSMV:00 and BSMV:SbNAC9 under mock or drought-stress treatments. Error bars indicate SD of three independent experiments. * p < 0.05 by Student’s t-test. (E–G) SOD (E), POD (F), and CAT (G) activities in sorghum seedlings inoculated with BSMV:00 and BSMV:SbNAC9 under mock or drought-stress treatments. Error bars indicate SD of three independent experiments. * p < 0.05 by Student’s t-test. (H) DAB and NBT staining of leaves of sorghum seedlings inoculated with BSMV:00 and BSMV:SbNAC9 under drought-stress treatment for 5 days. Bars indicate 5 cm.

Figure 5

SbNAC9 directly activated the expression of SbC5YQ75 and SbNCED3. (A,B) Relative transcript level of SbNAC9, SbNCED3, SbNCED9 (A), and C5YQ75 (B) in the third leaf of sorghum seedlings with silenced SbNAC9. SbEIF4A was used as the internal reference. Error bars indicate SD of three independent experiments. **** p < 0.0001, *** p < 0.001, and ** p < 0.01 by Student’s t-test. (C,D) EMSA assays of SbNAC9 bound to the promoters of SbC5YQ75 and SbNCED3 in vitro. Competition experiments were performed with excessive amounts of unlabeled probes (20× and 50× for SbC5YQ75 as well as 100× and 200× for SbNCED3). Schematic diagrams of putative binding motifs of SbNAC9 on the promoters of SbC5YQ75 and SbNCED3 were listed at the top. The motif at −264 bp upstream of transcription start sites of SbC5YQ75 and the motif at −838 bp upstream of transcription start sites of SbNCED3 were used for EMSA assays. The sequences of probes were listed at the bottom. (E) Schematic diagrams of constructs used in luciferase assays. (F) Relative transcript level of LUC/REN activity normalized by REN in luciferase assays. Error bars indicate SD of three independent experiments. ** p < 0.01 and * p < 0.05 by Student’s t-test. (G) Luciferase assays of SbNAC9 binding on SbC5YQ75 and SbNCED3 promoters in tobacco leaves. pSbC5YQ75:LUC or pSbNCED3:LUC with an empty vector were used as negative control.

Figure 6

The function of SbC5YQ75 in response to drought stress in sorghum. (A) Phenotype of sorghum seedlings inoculated with BSMV:00 and BSMV:SbC5YQ75 under mock and drought-stress treatments. Bars indicate 6 cm. (B) DAB and NBT staining of leaves of sorghum seedlings inoculated with BSMV:00 and BSMV:SbC5YQ75 treated with drought stress for 5 days. Bars indicate 5 cm. (C) Relative transcript level of SbC5YQ75 in sorghum seedlings silenced by VIGS. SbEIF4A was used as the internal reference. Error bars indicate SD of three independent experiments. ** p < 0.01 by Student’s t-test. (D–F) POD and (D) SOD (E) activities and MDA content (F) in sorghum seedlings inoculated with BSMV:00 and BSMV:SbC5YQ75 under mock or drought-stress treatments. Error bars indicate SD of three independent experiments. * p < 0.05 by Student’s t-test.

Figure 7

Model for SbNAC9 function in drought-stress tolerance of sorghum. Drought stress induces SbNAC9 expression. SbNAC9 directly activates the expression of SbC5YQ75 and SbNCED3 by binding to their promoters. In addition, SbNCED3 may potentially promote biosynthesis of ABA, which can induce SbNAC9 expression to form a feedback regulation. Additionally, SbNAC9 overexpression enhances drought-stress tolerance of sorghum through altering root architecture and increasing ROS scavenging ability.