CRISPR/Cas9 Mediated Knockout of the OsbHLH024 Transcription Factor Improves Salt Stress Resistance in Rice (Oryza sativa L.)

Abstract

Salinity stress is one of the most prominent abiotic stresses that negatively affect crop production. Transcription factors (TFs) are involved in the absorption, transport, or compartmentation of sodium (Na⁺) or potassium (K⁺) to resist salt stress. The basic helix-loop-helix (bHLH) is a TF gene family critical for plant growth and stress responses, including salinity. Herein, we used the CRISPR/Cas9 strategy to generate the gene editing mutant to investigate the role of OsbHLH024 in rice under salt stress. The A nucleotide base deletion was identified in the osbhlh024 mutant (A91). Exposure of the A91 under salt stress resulted in a significant increase in the shoot weight, the total chlorophyll content, and the chlorophyll fluorescence. Moreover, high antioxidant activities coincided with less reactive oxygen species (ROS) and stabilized levels of MDA in the A91. This better control of oxidative stress was accompanied by fewer Na⁺ but more K⁺, and a balanced level of Ca²⁺, Zn²⁺, and Mg²⁺ in the shoot and root of the A91, allowing it to withstand salt stress. Furthermore, the A91 also presented a significantly up-regulated expression of the ion transporter genes (OsHKT1;3, OsHAK7, and OsSOS1) in the shoot when exposed to salt stress. These findings imply that the OsbHLH024 might play the role of a negative regulator of salt stress, which will help to understand better the molecular basis of rice production improvement under salt stress.

Keywords: CRISPR/Cas9; OsbHLH024; ROS; antioxidants; rice; salt stress.

Figure 1

The expression level of OsbHLH024 in eight different tissues. OsUBQ5 was used as the control; three biological replicates were applied, and the values were expressed as means ± SE.

Figure 2

CRISPR/Cas9-induced mutation in the OsbHLH024 (LOC_Os01g39330) gene. (a) Schematic diagram of gene structure and two CRISPR/Cas9 target locations, with UTRs, exons, and introns and bHLH domain shown by blank rectangles, black rectangles, black lines, and dotted rectangles, respectively. The 20-nt target sequences are shown at the bottom of the gene structure; (b) DNA sequencing and alignments with WT identify the editing genotypes; the deletion is indicated by the red dash; (c) the expected protein structure of WT and A91. The dotted and grey showed the bHLH domain in WT and frameshift or premature stop in A91.

Figure 3

The phenotypic comparison of the A91 and WT. (a) The height of seedlings at one-week-old growing in ½ MS media; (b) the comparison of the root number, the root length and the shoot length in WT and A91 (n = 9 biological replicates); (c) the morphology of plants at the reproductive stage; (d) the measurement of plant height (n = 30 biological replicates); (e) the phenotype of tiller; (f) the measurement of internode length (n = 30 biological replicates); (g) the structure of panicle; (h) 10 seeds length; (i) 10 seeds width, values are expressed as means ± SE; * and ** denote significant t-test results at p < 0.05 and p < 0.01, respectively.

Figure 4

The growth characteristics of the A91 and WT exposed to salt stress treatments (0 and 150 mM NaCl). (a) The growth of 21-day-old seedlings before salt stress; (b) the growth of 21-day-old seedlings after 12 h stress; (c) the growth of 21-day-old seedlings after 4 days of stress; (d) 7 days recovery after 7 days of salt stress; (e) the survival rate after 7 days of recovery; (f) the fresh shoot weight after 7 days of stress; (g) total chlorophyll content; (h) SPAD value; (i) fluorescence; n = 3 biological replicates (e,f, each replicate represented 20 seedlings); 3 biological replicates (g), 9 biological replicates (h,i); data represented as means ± SE; ** indicates significant t-test results at p < 0.01; scale bar 10 cm.

Figure 5

The oxidative stress in the shoot and root of WT and A91 seedlings under salt stress. (a–d) activities of SOD, POD, H₂O_2, and MDA in shoot; (e–h) activities of SOD, POD, H₂O_2, and MDA in the root; (i) NBT staining (indicates O₂^•−); (j) DAB staining (indicates H₂O₂); n = 3 biological replicates, data represent means ± SE; * and ** indicate significant t-test results at p < 0.05 and p < 0.01, respectively.

Figure 6

Measurement of nutrient elements in the shoots and roots of WT and A91 seedlings exposed to salt stress for 7 days. (a–f) Na⁺, K⁺, Na⁺/K⁺, Ca²⁺, Mg²⁺, and Zn²⁺ in shoots; (g–l) Na⁺, K⁺, Na⁺/K⁺, Ca²⁺, Mg²⁺, and Zn²⁺ in roots; n = 3 biological replicates; data represent means ± SE; * and ** indicate significant t-test results at p < 0.05 and p < 0.01, respectively.

Figure 7

Expression of genes in WT and A91 under control and salt stress for 2 days. (a–f) OsHKT1;1, OsHKT1;3, OsHKT1;5, OsHAK7, OsSOS1 and OsLEA3 in shoots; (g–l) OsHKT1;1, OsHKT1;3, OsHKT1;5, OsHAK7, OsSOS1 and OsLEA3 in roots; OsACTIN1 was used as the control; n = 3 biological replicates, data represent means ± SE; * and ** indicate significant t-test results at p < 0.05 and p < 0.01, respectively.