OsSKL2 is a Chloroplast-Localized Protein Induced by Several Abiotic Stresses

In previous transcriptome data analysis of rice under high salinity treatment, we identified OsSKL2 and showed its high similarity to AtSKL2 (Additional file 1: Fig. S1). Since plant SK and SK-like homologs (SKLs) have never been reported in response to abiotic stress, this prompted us to investigate the function of OsSKL2 under stress conditions. First, the subcellular localization of OsSKL2 was analyzed by transforming rice leaf protoplasts with GFP-fused OsSKL2. In the control GFP protoplast, fluorescence was detected both in the cytoplasm and nucleus, while the OsSKL2-GFP fusion signal was observed only in the chloroplast (Fig. 1A), suggesting chloroplast localization. To further reveal the function of OsSKL2, qRT-PCR was performed to determine the transcriptional level of OsSKL2 in rice leaf, stem and root samples following high salinity, mannitol, H2O2 and ABA treatments (Fig. 1B). No significant difference of OsSKL2 expression was detected under normal condition (Additional file 1: Fig. S2). However, transcription levels were rapidly induced in the leaves following 1–3 h treatment with NaCl, PEG, H2O2 and ABA. In addition, varying transcriptional levels were detected in the stems and roots, with notably high levels following NaCl and ABA treatment. These results suggest that OsSKL2 is responsive to multiple abiotic stresses.

Fig. 1
figure 1

OsSKL2 subcellular localization and expression profiles. A Subcellular localization of OsSKL2 using a rice protoplast transient transformation system, showing chlorophyll localization (bar = 10 μm). B Inducible expression profiles of OsSKL2 in response to various abiotic stresses. Two-week-old rice seedlings (Zhonghua11) were treated with 100 mM NaCl, 100 mM mannitol, 5 mM H2O2, or 100 µM ABA. Root, stem and leaf samples were then harvested at 0, 1, 3, 9, 12, and 24 h after treatment, respectively. Expression levels of OsSKL2 were then determined with qRT-PCR using rice Actin1 as an internal control

OsSKL2 Positively Regulates Salt and Drought Tolerance in Rice

To explore the role of OsSKL2 in rice, we generated OsSKL2 overexpressing (OE) and RNAi transgenic (ZH11 background) lines. Two independent OsSKL2-OE (OE3 and OE6) and OsSKL2-RNAi lines (Ri6 and Ri9) possessing different transcription levels were confirmed by qRT-PCR and selected for the following research (Additional file 1: Fig. S3). Since OsSKL2 is a SK homolog (Fucile et al. 2008), we first chose to examine shikimate levels in the transgenic plants. Accordingly, compared with the wild-type (WT) plants, no obvious differences in the shikimate content were detected (Additional file 1: Fig. S4), suggesting that OsSKL2 does not possess encoded SK enzyme activity.

To confirm the function of OsSKL2 in salt and drought resistance in rice, we therefore examined the performance of WT, OsSKL2-OE and OsSKL2-RNAi seedlings on half-strength MS medium under high salinity (120 and 150 mM) and mannitol treatment (200 and 250 mM) at the germination stage. Under control conditions, with no NaCl or mannitol stress, no obvious differences were observed between the WT and transgenic plants. However, after 12 d of respective NaCl and mannitol treatment, the OsSKL2-OE lines exhibited higher relative shoot growth and a greater seminal root number than the WT seedlings. In contrast, the OsSKL2-RNAi lines showed lower relative shoot growth and a lower seminal root number than the WT (Additional file 1: Fig. S5). These results indicate that OsSKL2 plays a positive regulatory role during osmotic stress in rice at the germination stage.

Two-week-old rice seedlings of transgenic and WT plants grown in liquid Hoagland solution were subsequently treated with NaCl to create stress conditions. Under control conditions, no obvious differences were observed between any of the tested lines (Fig. 2A). However, after treatment with 120 mM NaCl for 10 d, the survival rate of the OsSKL2-OE lines was approximately twofold higher than that of the WT plants (Fig. 2B). In contrast, after 8-d treatment with 120 mM NaCl, the survival rate of the OsSKL2-RNAi lines was approximately twofold lower than that of the WT plants (Fig. 2B). A similar trend was also found between the transgenic and WT plants after treatment with 140 mM NaCl (Fig. 2C). In addition, ion leakage and the relative water content (RWC) were also measured in all lines following NaCl treatment. Compared with the WT, salt stress resulted in a significant decrease in relative ion leakage in the OsSKL2-OE plants, but a dramatic increase in the OsSKL2-RNAi plants (Fig. 2D). Meanwhile, the RWC of the OsSKL2-OE plants was significantly higher than that of the WT plants, while that of the OsSKL2-RNAi plants was remarkably lower (Fig. 2E). Next, growth of four-week-old transgenic and WT plants in soil was observed. Compared with the WT, the OsSKL2-OE plants exhibited a higher survival rate and RWC following treatment with 1.5% NaCl, while the OsSKL2-RNAi plants showed a lower survival rate and RWC (Additional file 1: Fig. S6). Taken together, these results demonstrate that OsSKL2 is crucial for enhancing salt tolerance in rice.

Fig. 2
figure 2

OsSKL2 positively regulates salt tolerance in rice. A Phenotypes of wild-type (WT), OsSKL2-overexpressing (OE, OE3 and OE6), and RNA interference (RNAi, Ri6 and Ri9) plants before (upper) and after treatment with 120 or 140 mM NaCl (middle and lower images). Two-week-old seedlings were used for salt treatment (bar = 5 cm). Survival rates of WT and OsSKL2 transgenic plants under B 120 and C 140 mM NaCl stress. Data represent means ± SD (n = 30). D Relative ion leakage and E the relative water content of WT and OsSKL2 transgenic plants under 120 and 140 mM NaCl stress. Data represent means ± SD. Three independent experiments were carried out with similar results. All data were analyzed using one-way analysis of variance (ANOVA) based on the Student’s t-test. *P < 0.05, **P < 0.01

The function of OsSKL2 during drought stress was also investigated in liquid Hoagland solution and soil. Under control conditions, no obvious differences were observed between the WT and transgenic lines (Fig. 3A). However, following treatment with 20% PEG for 10 d, the survival rate of the OsSKL2-OE lines was approximately 2.5-fold higher than that of the WT plants (Fig. 3B). In contrast, the survival rate of the OsSKL2-RNAi lines was approximately fourfold lower than that of the WT plants after treatment with 20% PEG for 8 d (Fig. 3B). A similar trend was also found between the transgenic lines and WT plants after treatment with 25% PEG (Fig. 3C). In addition, the RWC of the OsSKL2-OE plants was significantly higher, while that of the OsSKL2-RNAi plants was remarkably lower than the WT (Fig. 3D). Compared with the WT plants, PEG stress also resulted in a significant decrease in relative ion leakage in the OsSKL2-OE plants, but a dramatic increase in the OsSKL2-RNAi plants (Fig. 3E). Four-week-old transgenic and WT plants were subsequently planted in soil. After stopping irrigation then 2-d recovery, the OsSKL2-OE plants exhibited a higher survival rate, while the OsSKL2-RNAi plants presented a lower survival rate compared with the WT (Additional file 1: Fig. S7A & B). In addition, the OsSKL2-OE plants presented a higher RWC and the OsSKL2-RNAi plants a lower RWC compared with the WT (Additional file 1: Fig. S7C). Collectively, these results demonstrate that OsSKL2 is also important for improving drought tolerance in rice.

Fig. 3
figure 3

OsSKL2 positively regulates drought tolerance in rice. A Phenotypes of wild-type (WT), OsSKL2-overexpressing (OE, OE3 and OE6), and RNA interference (RNAi, Ri6 and Ri9) plants before (upper) and after treatment with 20 or25% PEG (middle and lower). Two-week-old seedlings were used for drought treatment (bar = 5 cm). Survival rates of WT and OsSKL2 transgenic plants under B 20 and C 25% PEG stress. Data represent means ± SD (n = 30). D Relative water contents and E relative ion leakage in WT and OsSKL2 transgenic plants under 20 and 25% PEG stress. Data represent means ± SD. Three independent experiments were carried out with similar results. All data were analyzed using one-way analysis of variance (ANOVA) based on the Student’s t-test. *P < 0.05, **P < 0.01

OsSKL2 Improves Oxidative Stress Tolerance in Transgenic Rice

Drought and high salinity can induce the production and accumulation of ROS (Foreman et al. 2003). Here, transcriptional levels of OsSKL2 were rapidly induced following H2O2 treatment (Fig. 1B), implying that OsSKL2 plays an important role in oxidative stress. To analyze the role of OsSKL2 in ROS scavenging, we therefore measured the H2O2 content of transgenic plants under drought and salt stress conditions. Following treatment, the OsSKL2-OE lines showed a lower H2O2 level, while the OsSKL2-RNAi lines displayed a higher H2O2 level than the WT (Fig. 4A). Additionally, several physiological indices related to antioxidant capacity and stress tolerance were also examined. Compared with the WT plants, the OsSKL2-OE lines showed a lower MDA content, and higher SOD, POD and CAT activities, while the OsSKL2-RNAi lines showed a higher MDA content, and lower activities of SOD, POD and CAT under drought and salt conditions (Fig. 4B–E). H2O2 levels in the WT and transgenic seedlings were subsequently confirmed by 3,3-diaminobenzidine (DAB) staining. Compared with the WT, weaker staining intensity was observed in the leaves and roots of the OsSKL2-OE lines, while the OsSKL2-RNAi lines showed stronger staining (Fig. 4F), suggesting an increase in H2O2 content. Exogenous H2O2 was subsequently used to investigate the responses of the OsSKL2 transgenic lines to oxidative stress. In the absence of H2O2, no differences in shoot height or root development were observed between the transgenic lines and WT. However, after exposure to 100 mM H2O2 for 10 d, while the OsSKL2-OE shoots and roots displayed superior growth, development was remarkably suppressed in the OsSKL2-RNAi lines compared with the WT (Fig. 4G–I). Taken together, these results suggest that OsSKL2 functions to maintain ROS homeostasis in rice.

Fig. 4
figure 4

OsSKL2 promotes tolerance to salt and drought stress by regulating H2O2 homeostasis in rice. Leaves of four-leaf-stage wild-type (WT) and OsSKL2 transgenic seedlings were treated with 25% PEG for 5 d or 140 mM NaCl for 7 d then measured for A H2O2, B MDA, C SOD, D POD and E CAT enzyme activity. Data represent means ± SD. F H2O2 histochemical analyses of 10-day-old seedlings treated with 250 mM mannitol or 140 mM NaCl then stained with 1% 3,3’-diaminobenzidine tetrachloride (DAB) (bar = 1 cm). G Phenotypes of WT and OsSKL2 transgenic plants before (upper) and after treatment with 100 mM H2O2 for 10 d (lower) (bar = 5 cm). H Seedling height and I the seminal root number of WT and OsSKL2 transgenic plants under 100 mM H2O2 stress. Data represent means ± SD (n = 30). Three independent experiments were carried out with similar results. All data were analyzed using one-way analysis of variance (ANOVA) based on the Student’s t-test. *P < 0.05, **P < 0.01

OsSKL2 Elevates ABA Sensitivity in Rice

ABA serves as an endogenous messenger during stress responses, with plant drought and salt responses closely related to ABA sensitivity (Yoshida et al. 2014). To determine whether OsSKL2 is involved in ABA responses, seedling height and root development were examined in the WT and transgenic lines following treatment with exogenous ABA for 10 d. Compared with the WT, significantly reduced shoot and root growth was observed in the OsSKL2-OE lines (Fig. 5A–C), suggesting that growth was arrested by ABA. In contrast, the OsSKL2-RNAi lines exhibited greater shoot growth and a higher root number compared with the WT (Fig. 5A–C), suggesting that less sensitive to ABA. Collectively, these results demonstrate that OsSKL2 increases sensitivity to ABA, highlighting an important role in the ABA response in rice.

Fig. 5
figure 5

OsSKL2 increases ABA sensitivity in rice. A Phenotypes of WT and OsSKL2 transgenic seedlings grown in 1/2 MS medium with or without 5/10 μM ABA for 10 d (bar = 5 cm). B Seedling height and C the seminal root number of WT and OsSKL2 transgenic plants under 5 and 10 μM ABA treatment. Data represent means ± SD (n = 30). Three independent experiments were carried out with similar results. All data were analyzed using one-way analysis of variance (ANOVA) based on the Student’s t-test. *P < 0.05, **P < 0.01

Since ABA induces ROS production (Watkins et al. 2017), we used DAB staining to examine whether exogenous ABA also affected ROS levels in the OsSKL2 transgenic lines. Under control treatment, there were no obvious differences in ROS accumulation between the WT and OsSKL2 transgenic lines. However, following treatment with 5 μM ABA for 10 d, strong DAB staining was observed in the leaves of the OsSKL2-OE lines, with weak staining in the OsSKL2-RNAi lines (Additional file 1: Fig. S8). These results suggest that the improved oxidative capability induced by OsSKL2 is mediated via the ABA regulatory pathway.

OsSKL2 Interacts With OsASR1

To determine the mechanism by which OsSKL2 modulates drought and salt tolerance, yeast two hybrid screening was performed using full-length OsSKL2 as bait. Several positive clones were obtained and found to correspond to the Abscisic Acid-Stress-Ripening (ASR) protein OsASR1. To verify this interaction, OsSKL2 and OsASR1 were co-transformed into yeast and verified on QDO medium. As expected, yeast containing the OsSKL2 and OsASR1 constructs grew well on QDO medium (Fig. 6A), suggesting interaction between OsSKL2 and OsASR1. To determine the regions of OsSKL2 that interact with the OsASR1 protein, full-length and fragmented OsSKL2 (OsSKL2-N and OsSKL2-C) were examined. As a result, OsSKL2 and OsSKL2-N were found to interact with OsASR1 (Additional file 1: Fig. S9A). Additionally, no interactions between other SK family members (OsSK1, OsSK2, OsSK3 and OsSKL1) and OsASR1 were observed (Additional file 1: Fig. S9B).

Fig. 6
figure 6

OsSKL2 interacts with OsASR1. A OsSKL2 was found to interact with OsASR1 in a yeast two-hybrid system, using BD-OsSKL2 + AD, AD-OsASR1 + BD as negative controls. Growth on selective QDO plates indicated a positive interaction. DDO: yeast SD/-Trp/-Leu selective medium, QDO: yeast SD/-Trp/-Leu/-His/-Ade selective medium. B A pull-down assay was then performed to confirm the interaction between OsSKL2 and OsASR1 in vivo. GST and GST-OsASE1 proteins coupled to beads were incubated with MBP-OsSKL2 fusion proteins. Bound OsSKL2 was then detected using mouse anti-MBP antibody. C A BIFC assay further revealed that OsSKL2 interacts with OsASR1 in rice leaf protoplasts (bar = 10 μm)

To confirm the interaction between OsSKL2 and OsASR1, recombinant proteins were purified in E. coli then used in a pull-down assay in vitro. The recombinant OsASR1-GST protein, but not GST alone, was able to pull down the OsSKL2 protein (Fig. 6B), implying a direct interaction between OsSKL2 and OsASR1. Furthermore, bimolecular fluorescence complementation (BiFC) assay was used to further confirm the interaction between OsSKL2 and OsASR1 in planta. The BiFC results showed that OsSKL2 specifically interacted with OsASR1, and the YFP signals were detected only following OsSKL2-YFPN/OsASR1-YFPC co-transfection of rice protoplasts localized to the nucleus (Fig. 6C). Collectively, these results confirm a direct interaction between OsSKL2 and OsASR1.

OsASR1 Plays an Important Role in Regulating Salt and Drought Tolerance in Rice

Previous research suggests that ASR genes either encode transcription factors or possess chaperone-like activity (Konrad and Bar-Zvi 2008; Arenhart et al. 2014; Li et al. 2017, 2018). To examine this, we characterized the subcellular localization and transcription activation of OsASR1. Both cytosol and nuclear localization of OsASR1 were detected in rice leaf protoplasts (Additional file 1: Fig. S10A). In addition, OsASR1 possessed no transcription activation activity in yeast (Additional file 1: Fig. S10B). These results suggest that OsASR1 does not possess the typical features of transcription factors.

Since OsASR1 interacts with OsSKL2, we also examined whether OsASR1 plays a pivotal role in salt and drought stress. To do so, we constructed OsASR1 OE and RNAi transgenic vectors (Additional file 1: Fig. S11A) then transformed them into WT rice (ZH 11). Several transgenic lines with differing transcription levels were obtained (Additional file 1: Fig. S11B-D) as confirmed by RT-PCR and qRT-PCR. Moreover, since the rice genome contains six ASR gene family members, we also examined the transcription levels of other rice ASR genes in the OsASR1RNAi lines. As a result, no remarkable reduction in these other ASR genes was observed in OsASR1RNAi5 (Ri5) and OsASR1RNAi7 (Ri7) compared with the WT (Additional file 1: Fig. S11E), suggesting that these plants were genuine OsASR1 knockdown lines. The Ri5 and Ri7 lines, and two OsASR1-OE (OE11 and OE12) lines were therefore selected for the following research.

To explore the role of OsASR1 in salt and drought stress, two-week-old seedlings of transgenic and WT plants grown in liquid Hoagland solution were treated with NaCl or PEG. Under control conditions, there were no obvious differences between the transgenic and WT plants (Fig. 7). However, after 10 d treatment with 120 mM NaCl, the OsASR1-OE lines were more robust and had a higher survival rate (58–67%) than the WT plants (32%). Conversely, after 8 d treatment with 120 mM NaCl, the OsASR1-RNAi plants were more wilted and had a lower survival rate (12—17%) than the WT plants (41%) (Fig. 7A, B). Consistent results were also observed between the transgenic and WT plants under 140 mM NaCl treatment (Fig. 7A, B). Meanwhile, under drought conditions, following 6 d treatment with 20% PEG, the WT plants exhibited more severe wilting and a yellowing phenotype than the OsASR1OE lines. Conversely, after 5 d treatment with PEG, the OsASR1-RNAi plants were more severely wilted and had a rolling phenotype compared to the WT (Fig. 7C, D). Moreover, following rewatering, the average survival rates of the two OsASR1OE lines (54 and 57%) were much higher than that of the WT (31%), while those of the two OsASR1RNAi lines (27 and 29%) were much lower than the WT (52%) (Fig. 7C, D). Taken together, these results suggest that OsASR1 positively regulates salt and drought stress in rice.

Fig. 7
figure 7

OsASR1 positively regulates salt and drought tolerance in rice. A Phenotypes of wild-type (WT), OsASR1-overexpressing (OE, OE11 and OE12), and RNA interference (RNAi, Ri5 and Ri7) plants before (upper) and after treatment with 120 or 140 mM NaCl (middle and lower). Two-week-old seedlings were used for salt treatment (bar = 5 cm). B Survival rates of WT and OsASR1 transgenic plants before and after 120 and 140 mM NaCl stress. Data represent means ± SD (n = 30). C Phenotypes of WT and OsASR1 transgenic plants before (upper) and after treatment with 20% PEG (middle), and after rewatering for 2 d (bottom). Two-week-old seedlings were used for drought treatment (bar = 5 cm). D Survival rates of WT and OsASR1 transgenic plants under drought stress. Data represent means ± SD (n = 30). Three independent experiments were carried out with similar results. All data were analyzed using one-way analysis of variance (ANOVA) based on the Student’s t-test. *P < 0.05, **P < 0.01

Transient Co-expression of OsSKL2 with OsASR1 Decreased ROS Production

To explore the possible regulatory mechanism of OsSKL2 and OsASR1 in enhancing tolerance to salt and drought in rice, we carried out transient expression in leaves of Nicotiana benthamiana. Compared with leaves expressing an empty vector, strong staining was detected in leaves expressing OsSKL2, OsASR1, and OsSKL2 + OsASR1 (Fig. 8A). However, weaker staining was observed in areas co-expressing OsSKL2 + OsASR1 compared with individual expression (Fig. 8B). These results suggest that transient co-expression of OsSKL2 + OsASR1 caused a reduction in ROS production.

Fig. 8
figure 8

Co-expression of OsSKL2 and OsASR1 decreased ROS production. A Transient co-expression of OsSKL2 and OsASR1 in the leaves of Nicotiana benthamiana. DAB-stained tobacco leaves were transiently transformed with an empty vector (P35S), OsSKL2 (P35S-OsSKL2), OsASR1 (P35S-OsASR1), and OsSKL2 + OsASR1, respectively (bar = 1 cm). B H2O2 contents of transiently transformed tobacco leaves.Three independent experiments were carried out with similar results. All data were analyzed using one-way analysis of variance (ANOVA) based on the Student’s t-test. **P < 0.01

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