Functional genomic analysis of K+ related salt-responsive transporters in tolerant and sensitive genotypes of rice

Abstract

Introduction: Salinity is a complex environmental stress that affects the growth and production of rice worldwide. But there are some rice landraces in coastal regions that can survive in presence of highly saline conditions. An understanding of the molecular attributes contributing to the salinity tolerance of these genotypes is important for developing salt-tolerant high yielding modern genotypes to ensure food security. Therefore, we investigated the role and functional differences of two K⁺ salt-responsive transporters. These are OsTPKa or Vacuolar two-pore potassium channel and OsHAK_like or a hypothetical protein of the HAK family. These transporters were selected from previously identified QTLs from the tolerant rice landrace genotype (Horkuch) and sensitive genotype (IR29).

Methods: In silico comparative sequence analysis of the promoter sequences of two these genes between Horkuch and IR29 was done. Real-Time expression of the selected genes in leaves and roots of IR29 (salt-sensitive), I-14 and I-71 (Recombinant Inbred Lines of IR29(♀)× Horkuch), Horkuch and Pokkali (salt-tolerant) under salt-stress at different time points was analyzed. For further insight, OsTPKa and OsHAK_like were chosen for loss-of-function genomic analysis in Horkuch using the CRISPR/Cas9 tool. Furthermore, OsTPKa was chosen for cloning into a sensitive variety by Gateway technology to observe the effect of gain-of-function.

Results: The promoter sequences of the OsTPKa and OsHAK_like genes showed some significant differences in promoter sequences which may give a survival advantage to Horkuch under salt-stress. These two genes were also found to be overexpressed in tolerant varieties (Horkuch and Pokkali). Moreover, a coordinated expression pattern between these two genes was observed in tolerant Horkuch under salt-stress. Independently transformed plants where the expression of these genes was significantly lowered, performed poorly in physiological tests for salinity tolerance. On the other hand, positively transformed T₀ plants with the OsTPKa gene from Horkuch consistently showed growth advantage under both control and salt stress.

Discussion: The poor performance of the transgenic plants with the down-regulated genes OsTPKa and OsHAK_like under salt stress supports the assumption that OsTPKa and OsHAK_like play important roles in defending the rice landrace Horkuch against salt stress, minimizing salt injury, and maintaining plant growth. Moreover, the growth advantage provided by overexpression of the vacuolar OsTPKa K⁺ transporter, particularly under salt stress reconfirms its important role in providing salt tolerance. The QTL locus from Horkuch containing these two transporters maybe bred into commercial rice to produce high-yielding salt tolerant rice.

Keywords: CRISPR/Cas9; Horkuch; OsHAK_like; OsTPKa; expression analysis; rice; salt-sensitive; salt-tolerant.

Figure 1

Differential expression analysis of OsTPKa and OsHAK_like by Real-Time quantitative PCR. (A, B) tissue specific expression analysis in IR29, Horkuch, I-14, I-71 and Pokkali at 24 hour under 150 mM salt-stress; (C, D) Salt-responsive genotype specific expression analysis in root of IR29 and Horkuch at 12 hour, 24 hour, 36 hour, 48 hour and 72 hour under 150 mM stress condition.

Figure 2

Coordinated expression analysis of OsTPKa and OsHAK_like in Horkuch (A) and IR29 (B).

Figure 3

Schematic diagram of the proposed mechanism of maintaining K+/Na+ ratio in cytoplasm by coordinated function of OsTPKa and OsHAK_like. (A) Initially under salt stress condition, (A) there is influx of Na+ in cell, (B) which ultimately leads to membrane depolarization, (C) Depolarized membrane inhibits the function of HAK transporter, (D) Vacuolar K+ releases in cytoplasm by TPKa channel, (E) it helps to inhibit the decrease of K+ in cytoplasm. (B) at a later stage of salt stress when there is no depolarization of membrane, (F) HAK participates in uptaking K+ from soil and thus (G) helps to maintain K+ concentration in cytoplasm.

Figure 4

(A) Hygromycin resistance assay of T₀ transformants. The wild type became yellow, but the positive transgenic lines remain green in presence of hygromycin, (B) molecular confirmation of transformed plants by targeting Cas9 and hptII gene. The lines with the PCR bands are the positive transgenic lines, wild type showed no such bands; (C) Leaf disc senescence assay of flag leaves from both OsTPKa_sgRNA and OsHAK_like_sgRNA T₀ transformants of Horkuch. sgRNA transformed transgenic lines performed poorly compared to non-transformed wild type.

Figure 5

Phenotype of salt-tolerance in transformed lines. (A, B) The transformed and wild type seedlings growth at three leaf stage in hydroponics solution with 150 mM salt stress and no stress conditions for 9 days. (C, D) The comparison of seedling injury (SES score) under stress between wild type and both OsTPKa_sgRNA and OsHAK_like_sgRNA transformed plants. The stars in the graph indicates significant differences (***P < 0.001, **P < 0.01, *P < 0.1, Two way ANOVA test).

Figure 6

(A, B) Seed germination test under 100 mM salt condition. OsTPKa_sgRNA and OsHAK_like_sgRNA transformed Horkuch performed poorly under saline condition compared to their respective wild type; (C, D) Comparison of Shoot length of germinated seeds in seed germination test for both lines. The stars in the graph indicates significant differences (***P < 0.001, Two way ANOVA test).

Figure 7

Effect of salt stress on phenotypical characteristics of transgenic plants. (A–D) Percentage increase in electrolyte leakage and H₂O₂ content; In all the parameters wild type Horkuch performed better compared to sgRNA inserted transgenic lines. The stars in the graph indicates significant differences (***P < 0.001, **P < 0.01, Ordinary one way ANOVA test).

Figure 8

Effect of salt stress on phenotypical characteristics of transgenic plants. (A, B) Percentage increase in shoot length (C, D) Percentage reduction in root length. In all the parameters wild type Horkuch performed better compared to sgRNA inserted transgenic lines. The stars in the graph indicates significant differences (***P < 0.001, **P < 0.01, Ordinary one way ANOVA test).

Figure 9

Effect of salt stress on phenotypical characteristics of transgenic plants. (A, B) Percent reduction in chlorophyll content; (C, D) Percentage increase in Na⁺/K⁺ ratio in shoot; (E, F) Percentage increase in Na⁺/K⁺ ratio in root under 150 mM salt condition. In all the parameters wild type Horkuch performed better compared to sgRNA inserted transgenic lines. The stars in the graph indicates significant differences (***P < 0.001, **P < 0.01, Ordinary one way ANOVA test).

Figure 10

(A) The transgenic plants (line 39 and line 21) showed healthy appearance under 120 mM salt stress compared to the wild type plants. (B) Transgenic Line 39 showed significant (P<0.01) low SES score (high tolerance) than the WT. **P < 0.01, *P < 0.1.

Figure 11

(A) Percent Reduction of shoot length was less in the transgenic lines and the wild type plants showed significantly high reduction (p < 0.01) under stress compared to no stress. (Transgenic lines = 3, 21, 39, WT = Wild Type BRRI dhan28). (B) Percent increase in root length was higher in the transgenic lines and significantly higher (p < 0.05) in line 39 under salt stress compared to no stress and the wild type plants showed reduction under stress. (Transgenic lines = 3, 21, 39, WT = Wild Type BRRI dhan28). (C) Percent Reduction of shoot weight was less in the transgenic lines and the wild type plants showed significantly high (p<0.01) reduction under stress compared to without stress condition. (Transgenic lines = 3, 21, 39, WT = Wild Type BRRI dhan28).(D) Percent reduction in root weight was less in the transgenic lines and the wild type plants under 120 mM salt stress showed significant reduction (p < 0.05) compared to without stress condition. (Transgenic lines = 3, 21, 39, WT = Wild Type BRRI dhan28). (E) Significant increase ( p < 0.05) in K+/Na+ ratio was observed in the shoot tissues of transgenic lines 3, 21 and 39 under salt stress compared to their without stress condition which was not observed in the wildtype. (F) Significant decrease (p < 0.05) in K+/Na+ ratio was observed in root in the wild type compared to no stress. Transgenic line 39 showed higher increase under salt stress. (Transgenic lines = 3, 21, 39, WT = Wild Type BRRI dhan28). **P < 0.01, *P < 0.1.