A novel gene OsAHL1 improves both drought avoidance and drought tolerance in rice

Abstract

A novel gene, OsAHL1, containing an AT-hook motif and a PPC domain was identified through genome-wide profiling and analysis of mRNAs by comparing the microarray of drought-challenged versus normally watered rice. The results indicated OsAHL1 has both drought avoidance and drought tolerance that could greatly improve drought resistance of the rice plant. Overexpression of OsAHL1 enhanced multiple stress tolerances in rice plants during both seedling and panicle development stages. Functional studies revealed that OsAHL1 regulates root development under drought condition to enhance drought avoidance, participates in oxidative stress response and also regulates the content of chlorophyll in rice leaves. OsAHL1 specifically binds to the A/T rich sequence region of promoters or introns, and hence directly regulates the expression of many stress related downstream genes.

Figure 1. Relative expression of OsIAHL1 in rice.

The relative expression of OsAHL1gene in cultivar IRAT109 (Oryza sativa L. ssp japonica) with the plant hormones (A–D), and dehydration (E–H). OsAHL1gene responded to plant hormones (A) ABA, (B) H₂O₂, (C) JA, (D) SA, and dehydration, such as (E) cold (4 °C), (F) Mannitol, (G) NaCl treatment in 4 leaf stage; and (H) under water withholding condition in different developmental stages. The plants grown in normal watering conditions were used as a control. Error bars are the SD based on 3 replicates.

Figure 2. Expression pattern of GFP driven by the OsAHL1 promoter (P_AHL1) in transgenic rice plants under normal conditions.

Panels on the left were taken under visible light and the right under UV. GFP signal was monitored in vascular bundles of leaf blade (A,B), leaf sheath (C,D), stem internode (E,F), stem node (G,H) and root (I,J).

Figure 3. Overexpression of OsAHL1 gene enhanced abiotic stress resistance of rice at the seedling stage.

In panel I, the survival rate of transgenic (I17 lines), WT and RNAi plants at the seedling stage was investigated under 20% PEG(I-A), 150 mM salt (I-B) and 4 °C (I-C) stresses. Figure panel II is the growth status of OsAHL1 transgenic lines, WT and RNAi plants. II-A and II-B were before and at the end of seedling test in 20% PEG600 respectively, while II-C and II-D were in 150 mM salt stress. The 4 leaf rice plants were transplanted in hydroponic culture medium and treated with the above stresses for 7 days, then returned to normal condition for another 7 days. Error bars are the SD based on 3 replicates; every replicate contains 48 individual plants. Data bars in panel I with no superscripts in common indicate significant difference at p < 0.01.

Figure 4. Over-expression of OsAHL1 gene improved water-content ability of rice leaves.

(A) The relative water content (RWC) of leaves under drought stress. (B) The rate of water loss for separated leaves. Error bars are the SD based on 3 replicates. *Indicates significant difference by the t-test at p < 0.05, compared to WT.

Figure 5. Overexpression of OsAHL1 improved drought resistance at reproductive stage.

(A) OsAHL1 overexpressed plants (I17 lines) and WT under well-watered conditions (a), and drought stress (b). (B) Agronomic traits under normal conditions and drought stress: (a) seed-setting rate, (b) grain yield per plant, (c) the biological yield per plant, and (d) the 100-seed grain weight. Error bars are the SD based on 4 (welled-water) or 8 (under drought) plant replicates. *Indicates significant difference by the t-test at p < 0.05, compared to WT; **Means significant difference at p < 0.01.

Figure 6. OsAHL1-Overexpression improved POD activity and regulated chlorophyll content in the leaves of rice.

(A) POD activity of I17 plant lines and WT under normal and drought conditions. (B) Comparison of leaf chlorophyll content (a), chlorophyll composition (b,c) and chlorophyll a/b ratio (d) between I17 plant lines and WT under normal and drought condition. Error bars are the SD based on 3 replicates. *Indicates significant difference by the t-test at p < 0.05, compared to WT plants under the same condition; ^#Means significant difference by the t-test at p < 0.05, comparison between control and drought stress for the same plant line.

Figure 7. OsAHL1 regulated the development of rice root system under drought conditions at the reproductive stage.

Root volume per plant between OsAHL1 overexpression (I17) plant lines (A) and OsAHL1-RNAi plant lines (B) compared with WT under normal and drought conditions. (a) Total root volume, (b) upper root volume, and (c) lower root volume. Error bars are the SD based on 3 replicates. *Indicates significant difference by the t-test at p < 0.05, compared to WT; **Means significant difference at p < 0.01.

Figure 8. The role of AT-hook and DUF296 on nuclear localization of OsAHL1.

The figure panels are confocal microscopic images of OsAHL1-EGFP fusion protein driven by CaMV35s promoter in onion epidermal cell. The EGFP driven by CaMV35s promoter was used as positive control. The left schematic diagrams represent the expressed proteins. The AT-hook motif was indicated by red line, and DUF296 was indicated by blue box.

Figure 9. Yeast Two-hybrid tested the homologous combination capability among OsAHL1 proteins (or its DUF296 domains).

(A) Vector construct for yeast expression of OsAHL1 protein. (B) OsAHL1 proteins or its DUF296 domains could be bound together in yeast stain YRG-2. Yeast were diluted and dotted on amino acid deficiency SD medium with 50 mM 3-AT.

Figure 10. OsAHL1 binds to the target genes directly.

Yeast One-Hybrid Assay was used to analyze the interactions between OsAHL1 protein and the DNA region of its candidate target genes. Yeast were diluted and dripped on amino acid deficiency SD medium with gradient 3-AT. The genes were labeled on the left, and “i” indicates the intron region, the others are the promoter region of the genes. (B) The consensus of DNA motifs that OsAHL1 protein interacts with are predicted by MEME.

Figure 11. EMSA results.

Electrophoretic mobility shift assay (EMSA) using oligonucleotide probes containing the OsAHL1 binding region. The **denotes unbound biotin labeled DNA, *denotes OsAHL1-DNA (gene LOC_Os02g33420) complex.

Figure 12. OsAHL1 enhanced the expression of the target genes and other drought- related genes in rice.

The relative expression of the target genes and other drought- related genes in the leaf and root of OsAHL1 overexpression lines and WT plants under normal condition was quantified by qPCR. Error bars are the SD based on 3 replicates.