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. 2020 Jun 11;21(11):4163.
doi: 10.3390/ijms21114163.

Ectopic Expression of OsPYL/RCAR7, an ABA Receptor Having Low Signaling Activity, Improves Drought Tolerance without Growth Defects in Rice

Nikita Bhatnagar  1 , Rigyeong Kim  1 , Seungsu Han  2 , Jaeeun Song  1 , Gang Seob Lee  3 , Sangho Lee  2 , Myung Ki Min  1 , Beom-Gi Kim  1
Affiliations

Ectopic Expression of OsPYL/RCAR7, an ABA Receptor Having Low Signaling Activity, Improves Drought Tolerance without Growth Defects in Rice

Nikita Bhatnagar et al. Int J Mol Sci. .

Abstract

Overexpression of abscisic acid (ABA) receptors has been reported to enhance drought tolerance, but also to cause stunted growth and decreased crop yield. Here, we constructed transgenic rice for all monomeric ABA receptors and observed that only transgenic rice over-expressing OsPYL/RCAR7 showed similar phenotype with wild type, without total yield loss when grown under normal growth condition in a paddy field. Even though transgenic rice over-expressing OsPYL/RCAR7 showed neither an ABA-sensitivity nor an osmotic stress tolerance in plate assay, it showed drought tolerance. We investigated the ABA-dependent interaction with OsPP2CAs and ABA signaling induction by OsPYL/RCAR7. In yeast two hybrid assay, OsPYL/RCAR7 required critically higher ABA concentrations to interact with OsPP2CAs than other ABA receptors, and co-immunoprecipitation assay showed strong interaction under ABA treatment. When ABA-responsive signaling activity was monitored using a transient expression system in rice protoplasts, OsPYL/RCAR7 had the lowest ABA-responsive signaling activity as compared with other ABA receptors. OsPYL/RCAR7 also showed weak suppression of phosphatase activity as compared with other ABA receptors in vitro. Transcriptome analysis of transgenic rice over-expressing OsPYL/RCAR7 suggested that only a few genes were induced similar to control under without exogenous ABA, but a large number of genes was induced under ABA treatment compared with control. We conclude that OsPYL/RCAR7 is a novel functional ABA receptor that has low ABA signaling activity and exhibits high ABA dependence. These results lay the foundation for a new strategy to improve drought stress tolerance without compromising crop growth.

Keywords: ABA signaling; abiotic stress signaling; crop; growth and stress tolerance balance.

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Conflict of interest statement

The authors declare no conflict of interests.

Figures

Figure 1
Figure 1
Agricultural traits of rice over-expressing OsPYL/RCAR7 in paddy field. (A) Rice plants grown in a paddy field. Image was recorded just before the harvest. (B–F) Graphical representation of quantified data of the major agricultural traits of rice productivity. Values represent the average and error bars depict standard deviation. Data were recorded from at least 20 individual plants per line. ** means p-value is less than 0.01. *** means p-value is less than 0.001. – means p-value is higher than 0.05.
Figure 2
Figure 2
Post-germination assay of transgenic rice lines overexpressing OsPYL/RCAR7 under abscisic acid (ABA) treatment and osmotic stress conditions. (A,C,E) Representative images of rice seedlings grown on media, 1/2MS, supplemented with 5 μM ABA and 200 mM mannitol. (B,D,F) Quantitiative data of the post-germination assay. No significant differences were detected. The graphs show average values with standard error (n = 5). Three indepent experiments were performed and showed similar results in every repeats. Representative results were presented. – means no significance.
Figure 3
Figure 3
Transgenic rice over-expressing OsPYL/RCAR7 showed drought tolerance in vegetative stage. (A) Representative images of plants grown in the drought tolerance assay. All plants over-expressing OsPYL/RCAR7 (C14s) had the higher survival rate than wild type (WT) (DJs). Upper panel represents plants before drought stress treatment. Lower panel represents plants after drought stress treatment. (B) Graphical representation of survival rate after drought stress. Survival rate was calculated by counting the percentage of surviving plants in each pot. (C) Graphical representation of quantified fresh weight after drought stress. The graphs show average values with standard error (n = 6). The assay was conducted in triplicate and showed similar results. Plants were subjected to drought treatment for 7–10 days before recommencing watering. ** means p-value is less than 0.01,*** means p-value is less than 0.001 and **** means p-value is less than 0.0001.
Figure 4
Figure 4
Comparative transcriptome analysis of transgenic rice overexpresing OsPYL/RCAR7 and OsPYL/RCAR3. (A) Total transcripts of C14 (transgenic plants over-expressing OsPYL/RCAR7) (blue) and A30 (transgenic plants over-expressing OsPYL/RCAR3) (black) treated with no ABA were compared with DJ (wild type). ‘r’ is pearson correlation coefficient. FC is fold change. (B) Induced or reduced transcripts in C14 (blue) or A30 (black) treated with ABA were compared. (C) Venn diagram of transcripts with more than twofold changes under ABA treatment. (D) Heat map of common trascripts (fc > 2). (E,F) Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis with ABA dependently induced gene. Each transcript was detected in treated samples with indicated ABA treatment for 12 h. Similar expression levels were observed in triplicates. Error bars are SD. **** means that p-value is less than 0.0001. FPKM, fragment per kilo base of transcript per million mapped reads.
Figure 5
Figure 5
OsPYL/RCAR7 has stringent ABA dependency to interact with OsPP2CAs. (A) Yeast two hybrid analysis using OsPYL/RCAR7 and OsPYL/RCAR5 as baits and OsPP2CAs as preys. Synthetic minimal medium lacking leucine, tryptophan, and histidine (SD-LTH) supplemented with 0 to 5 μM concentration of ABA were used. Photographs were taken 5 days after inoculation of transformed yeasts. (B) OsPYL/RCAR7-GFP (green fluorescent protein) was immunoprecipiated and co-immunoprecipiated OsPP2C51 was detected by human influenza hemagglutinin (HA) antibody. (C) Inhibition assay of phosphatase activity for OsPPC50 was performed by three ABA receptors (OsPYL/RCAR3, 7, and 10).
Figure 6
Figure 6
OsPYL/RCAR7 showed the lowest activity among monomeric ABA receptors. (A) Comparative analysis of ABA signaling effects of monomeric ABA receptors under absence or presence of ABA. Luciferase (LUC) assay was done using rice protoplasts, reporter, and ABA receptors as signaling effectors. (B,C) Comparative analysis of OsPYL/RCAR7 and OsPYL/RCAR5 activity to suppress individual phosphatase PP2C08 and OsPP2C30. Luciferase assay was done using rice protoplasts, reporter, OsPP2CAs, and ABA receptors. Luciferase activity was measured by comparing with co-transformed marker (Ubi::rLUC), fLUC/rLUC. Values represent averages and error bars are SD (n = 3). These experiments were repeated three times. Results showed similar patterns and representative results were presented. – means that p-value is more than 0.05, * means that p-value is less than 0.05, ** means that p-value is less than 0.01 and **** means that p-value is less than 0.0001.
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