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. 2023 Jun 28;24(13):10821.
doi: 10.3390/ijms241310821.

PROTEIN PHOSPHATASE 2C08, a Negative Regulator of Abscisic Acid Signaling, Promotes Internode Elongation in Rice

Jaeeun Song  1 , Eunji Ga  1 , Sangkyu Park  1 , Hyo Lee  1 , In Sun Yoon  2 , Saet Buyl Lee  1 , Jong-Yeol Lee  1 , Beom-Gi Kim  1
Affiliations

PROTEIN PHOSPHATASE 2C08, a Negative Regulator of Abscisic Acid Signaling, Promotes Internode Elongation in Rice

Jaeeun Song et al. Int J Mol Sci. .

Abstract

Clade A protein phosphatase 2Cs (PP2CAs) negatively regulate abscisic acid (ABA) signaling. Here, we investigated the functions of OsPP2CAs and their crosstalk with ABA and gibberellic acid (GA) signaling pathways in rice (Oryza sativa). Among the nine OsPP2CAs, OsPP2C08 had the highest amino acid sequence similarity with OsPP2C51, which positively regulates GA signaling in rice seed germination. However, OsPP2C08 was expressed in different tissues (internodes, sheaths, and flowers) compared to OsPP2C51, which was specifically expressed in seeds, and showed much stronger induction under abiotic stress than OsPP2C51. Transgenic rice lines overexpressing OsPP2C08 (OsPP2C08-OX) had a typical ABA-insensitive phenotype in a post-germination assay, indicating that OsPP2C08, as with other OsPP2CAs, negatively regulates ABA signaling. Furthermore, OsPP2C08-OX lines had longer stems than wild-type (WT) plants due to longer internodes, especially between the second and third nodes. Internode cells were also longer in OsPP2C08-OX lines than in the WT. As GA positively regulates plant growth, these results suggest that OsPP2C08 might positively regulate GA biosynthesis. Indeed, the expression levels of GA biosynthetic genes including gibberellin 20-oxidase (OsGA20ox4) and Ent-kaurenoic acid oxidase (OsKAO) were increased in OsPP2C08-OX lines, and we observed that GIBBERELLIN 2-OXIDASE 4 (OsGA2ox4), encoding an oxidase that catalyzes the 2-beta-hydroxylation of several biologically active GAs, was repressed in the OsPP2C08-OX lines based on a transcriptome deep sequencing and RT-qPCR analysis. Furthermore, we compared the accumulation of SLENDER RICE 1 (SLR1), a DELLA protein involved in GA signaling, in OsPP2C08-OX and WT plants, and observed lower levels of SLR1 in the OsPP2C08-OX lines than in the WT. Taken together, our results reveal that OsPP2C08 negatively regulates ABA signaling and positively regulates GA signaling in rice. Our study provides valuable insight into the molecular mechanisms underlying the crosstalk between GA and ABA signaling in rice.

Keywords: ABA signaling; GA biosynthesis; GA signaling; PP2CA.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
OsPP2C08 expression profile in different rice tissues and OsPP2C08 subcellular localization. (A) OsPP2C51 and OsPP2C08 expression in response to abiotic stresses. All values were determined by RT−qPCR and normalized to UBIQUTIN 5 levels using the ∆Ct method. (B) OsPP2C08 expression levels in shoots and roots of young rice seedlings by RT−qPCR and normalized to UBIQUTIN 5 levels, using the ∆Ct method (Student’s t-test and nonparametric test with unpaired test, ***: p < 0.001). (C) OsPP2C08 expression levels in mature rice tissues (leaf blade, leaf sheath, stem, tiller, and flower) by RT−qPCR and normalized to UBIQUTIN 5 levels, using the ∆Ct method (one-way ANOVA with Tukey’s test, ****: p < 0.0001). Data are means ± SEM (n = 3). (D) Histochemical GUS staining analysis of proOsPP2C08:GUS transgenic rice seedlings and plants. Two-day-old seedlings (a), two-week-old seedlings (b), nodes (c), longitudinal section of a node (white arrow indicates the intercalary meristem) (d), horizontal section of a node (e), leaf blade (f), lamina joint (g), flower (h), and panicle (i). (E) Subcellular localization of OsPP2C08−GFP in a rice protoplast. OsPP2C−GFP was used at 10 μg per transfection. Exposure time of GFP fluorescence was 200 ms. Scale bars, 10 μm.
Figure 2
Figure 2
ABA−insensitive phenotypes of transgenic rice overexpressing OsPP2C08. (A) OsPP2C08-OX(#01, #02, #04) and WT (Wild type) seedlings were transferred 3 days after germination to half-strength MS medium without ABA (top panel), with 1 μM ABA (middle panel) or with 5 μM ABA (bottom panel). Photographs show representative seedlings at 4 days (top panel) or 5 days (middle and bottom panel) after transfer. Scale bar, 1 cm. (B) Shoot growth rate (upper panel) and root growth rate (bottom panel) of OsPP2C08−OX and WT plants grown with or without ABA (n = 12). Data are means ± SEM. (one-way ANOVA with Tukey’s test, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001). (C) Relative OsRAB16A and OsLEA03 expression in OsPP2C08-OX seedlings at 6 h after 5 μM ABA treatment. Gene expression levels were determined by RT−qPCR, and normalized to UBIQUTIN 5 levels using the ∆∆Ct method. Data are means ± SEM (one-way ANOVA with Tukey’s test, ***: p < 0.001).
Figure 3
Figure 3
Internode elongation phenotypes in the OsPP2C08−OX lines. (A) Representative photograph of mature OsPP2C08-OX and WT plants grown in a paddy field. (B) Culm length, panicle length, number of panicles, and total seed weight of OsPP2C08-OX and WT plants (n = 7). Data are means ± SEM. (one-way ANOVA with Tukey’s test, ***: p < 0.001). (C) Representative photograph of rice stems in OsPP2C08-OX and WT plants grown in a paddy field. Scale bar, 10 cm. (D) Internode length in OsPP2C08-OX and WT plants (n = 7). Data are means ± SEM. Asterisks indicate a significant difference (one-way ANOVA with Tukey’s test, *: p < 0.05, **: p < 0.01 ***: p < 0.001). (E) Representative micrographs of cell walls in the first internode of OsPP2C08-OX and WT plants grown in a paddy field. Scale bar, 20 μm. (F) Cell length in OsPP2C08-OX and WT plants (n = 5). Data are means ± SEM (t-test and parametric test with paired test, ***: p < 0.001, ****: p < 0.0001).
Figure 4
Figure 4
GA−sensitive phenotype of the OsPP2C08-OX lines(#01, #02, #04), expression analysis of GA biosynthesis genes, and immunoblotting for SLR1. (A) OsPP2C08-OX and WT seedlings were transferred at 3 days after germination to half-strength MS medium containing 10 μM GA. Control (a), 10 μM GA (b). Representative photograph of seedlings 4 days after transfer. Scale bar, 2 cm. (B) Shoot growth rate (left panel) and root growth rate (right panel) of OsPP2C08-OX and WT seedlings grown with or without GA (n = 10). Data are means ± SEM. (one-way ANOVA with Tukey’s test, **: p < 0.01 ****: p < 0.0001). (C) Relative expression of GA biosynthesis genes in 7-day-old OsPP2C08-OX(#) and WT seedlings, as determined by RT−qPCR. Data are means ± SEM of 3 biological replicates. (one-way ANOVA with Tukey’s test, ****: p < 0.0001). (D) Immunoblotting for SLR1.
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