Salicylic acid-related ribosomal protein CaSLP improves drought and Pst.DC3000 tolerance in pepper

Abstract

The ribosomal protein contains complex structures that belong to polypeptide glycoprotein family, which are involved in plant growth and responses to various stresses. In this study, we found that capsicum annuum 40S ribosomal protein SA-like (CaSLP) was extensively accumulated in the cell nucleus and cell membrane, and the expression level of CaSLP was up-regulated by Salicylic acid (SA) and drought treatment. Significantly fewer peppers plants could withstand drought stress after CaSLP gene knockout. The transient expression of CaSLP leads to drought tolerance in pepper, and Arabidopsis's ability to withstand drought stress was greatly improved by overexpressing the CaSLP gene. Exogenous application of SA during spraying season enhanced drought tolerance. CaSLP-knockdown pepper plants demonstrated a decreased resistance of Pseudomonas syringae PV.tomato (Pst) DC3000 (Pst.DC3000), whereas ectopic expression of CaSLP increased the Pst.DC3000 stress resistance in Arabidopsis. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) results showed that CaNAC035 physically interacts with CaSLP in the cell nucleus. CaNAC035 was identified as an upstream partner of the CaPR1 promoter and activated transcription. Collectively the findings demonstrated that CaSLP plays an essential role in the regulation of drought and Pst.DC3000 stress resistance.

Keywords: CaSLP; Drought tolerance; Pepper; Salicylic acid; Stomatal.

Fig. 1

Subcellular localization of CaSLP and the expression pattern of CaSLP were induced by salicylic acid and drought. A Green fluorescent protein (GFP) control vector (35S:GFP) or CaSLP-GFP fusion protein (35S:CaSLP-YFP) was transiently expressed in Nicotiana benthamiana leaves. Fully automatic microscopic fluorescence images were acquired under green fluorescence and bright field. Scale bars, 50 μm. B CaSLP transcript levels after drought treatment. The pepper leaves were taken at the 0, 1, 3, 6, 12, and 24 h time points for transcript level analysis. C CaSLP transcript levels under SA treatment. Leaves were acquired at 0, 1, 3, 6, 12, and 24 h time points. Actin was chosen as a control. Error bars show ± SD (n = 3)

Fig. 2

Silencing of CaSLP by virus-induced gene silencing (VIGS) decreases drought tolerance in pepper. A Phenotype of CaSLP-silenced and control (TRV, tobacco rattle virus) plants before and after 15 d of drought treatment. Plants were treated under water deficit conditions for 15 days, then rewatered for 3 days. B, E Histochemical staining of 3,3’-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) was performed to detect the accumulation of H₂O₂ and O₂^.− levels, respectively. C, F H₂O₂ and O₂.^.− contents. G Chlorophyll contents of the CaSLP-silenced and control plants before and after 15 d of drought treatment. H Photomicrographs of stomata from the CaSLP-silenced and control plants. I The stomatal aperture was analyzed under the microscope. J Stomatal density K Water loss rate of the CaSLP-silenced and control plants. Error bars represent ± SD (n = 3). Asterisks represent a significant difference between CaSLP-silenced and control plants under the same condition (*, P < 0.05; **, P < 0.01)

Fig. 3

Transient expression of CaSLP significantly improved pepper drought tolerance. A The transcript levels of CaSLP. B Phenotype of CaSLP-To (Transient overexpression) and the control (35S::GFP) plants before and after 48 h of drought treatment. Plants were treated under water deficit conditions for 48 h. C-G Stomatal aperture (C), Water loss rate (D), H₂O₂ content (E), O₂.^.− content (F), Contents of CaSLP-To and the control plants before and after 48 h of drought treatment. G Stomatal aperture. H Stomatal density. I-K Transcript levels of the stomatal development genes SDD1 (H), YODA (I), and FAMA(J) were measured by qRT-PCR in CaSLP-To and the control plants before and after the drought treatment. Values are means ± SD (n = 3). Different asterisks represent significant differences (*, P < 0.05; **, P < 0.01)

Fig. 4

Overexpression of CaSLP confers enhanced drought tolerance to transgenic Arabidopsis. A-B The phenotype of transgenic and wild-type (WT) plants before and after 10 d of drought treatment. Plants were treated under water deficit conditions for 10 d, then rewatered for 3 d. C-E Relative electrolyte leakage (C), and Malondialdehyde (MDA) content (D), Chlorophyll content (E) of the transgenic and WT plants before and after the drought treatment. Values are means ± SD (n = 3). Asterisks show a significant difference between the transgenic lines and WT under drought stress (*, P < 0.05; **, P < 0.01)

Fig. 5

Exogenous spraying salicylic acid enhanced the drought tolerance of CaSLP-silenced plants. A Phenotypes of the VIGS line (TRV-CaSLP) and TRV control before and after exogenous spraying salicylic acid treatment. B-D Proline content (B), relative electrolyte leakage (C), and malondialdehyde (MDA) content (D) of the VIGS line (TRV-CaSLP) and TRV control plants. Values are means ± SD (n = 3 replicates). Asterisks represent a significant difference between the transgenic lines and WT under drought stress (*, P < 0.05; **, P < 0.01)

Fig. 6

CaSLP affects the expression levels of stomatal development genes. A-C The transcript levels of stomatal development-related genes, including SDD1, YODA, and FAMA were analyzed by qRT-PCR in VIGS line (TRV-CaSLP) and TRV control plants, D-H AtSDD1, AtYODA, AtFAMA, AtTMM, and AtMPK3 were analyzed by qRT-PCR in transgenic lines and wild-type (WT) plants. Values are means ± SD (n = 3 replicates). Asterisks indicate a significant difference between the transgenic lines and WT under drought stress (*, P < 0.05; **, P < 0.01)

Fig. 7

Silencing of CaSLP by virus-induced gene silencing (VIGS) decreases Pst.DC3000 resistance in pepper. A, E Disease symptoms of CaSLP-silenced and control (TRV, tobacco rattle virus) plants before and after the Pst.DC3000 infection. Plants were infected with Pst.DC3000 for 3 days. B, F Histochemical staining with 3,3’-diaminobenzidine (DAB) and Trypan blue for analyzing the accumulation of H₂O₂ in the CaSLP-silenced and control plants before and after 3 d of Pst.DC3000 infection. C, G Trypan blue and DAB staining for cell death in the CaSLP-silenced and control plants before and after Pst.DC3000 infection. Bar = 50 μm. D-I Bacterial numbers (D), Chlorophyll content (H), and H₂O₂ content (I) of the CaSLP-silenced and control plants before and after 3 d of Pst.DC3000 infection. J-L The expression of the SA response genes of CaNPR1, CaABR1 and CaPR1. M, O₂^.− content of the CaSLP-silenced and control plants before and after Pst.DC3000 infection. Error bars represent ± SD (n = 3). Asterisks indicate a significant difference between CaSLP-silenced and control plants(*, P < 0.05; **, P < 0.01)

Fig. 8

Overexpression of CaSLP confers enhanced resistance to Pst.DC3000 stress in transgenic Arabidopsis. A, D Phenotype of transgenic and WT plants after the Pst.DC3000 infection. Plants were infected with Pst.DC3000 for 3 days. B, E Histochemical staining with 3,3’-diaminobenzidine (DAB) and Trypan blue for measuring the H₂O₂ contents of the transgenic and WT plants after 3 d of Pst.DC3000 infection. Bar = 50 μm. C, F Trypan blue and DAB staining for cell death in the transgenic and wild-type (WT) plants after 3 d of Pst.DC3000 infection. G Bacterial numbers. H Chlorophyll contents in the transgenic and WT plants after 3 d of Pst.DC3000 infection. I-K The expressions of the SA response genes of AtNPR1, AtPAL3, and AtICS. Values are means ± SD (n = 3 replicates). Asterisks indicate a significant difference between the transgenic lines and WT under drought stress (*, P < 0.05; **, P < 0.01)

Fig. 9

CaSLP enhances the binding of CaNAC035 to its target gene promoters. A Yeast two-hybrid assay of CaNAC035 and CaSLP. The full-length ones were CaNAC035 and CaSLP cloned into a pGBKT7 or a pGADT7 vector, respectively Yeasts grown in SD (-Trp/-Leu), SD (-Trp/-Leu/-His/-Ade) and SD (-Trp/-Leu/-His/-Ade + X-α-gal) media are indicated. B Bimolecular fluorescence complementation (BiFC) assay of CaNAC035 and CaSLP. A representative picture is shown here, GFP, green fluorescent protein. C Growth of yeast cells. D, F Diagrams of effector and reporter constructs. E luciferase (LUC)/Renilla luciferase (REN) activities detected from LUC/REN reporter system. G, H GUS activities assay the interaction of CaSLP and enhance the binding of CaNAC035 to the CaPR1 promoter. Values are means ± SD (n = 3 replicates). Asterisks indicate significant difference (*, P < 0.05; **, P < 0.01)

Fig. 10

CaNAC035 is required for CaSLP-mediated drought stress tolerance. A Phenotypes of CaSLP-silenced cells were coagroinoculated into 35S:CaNAC035:GFP. B, C The transcript levels of CaNAC035 and CaSLP in TRV2:CaNAC035/35S:CaSLP:GFP, TRV2:00/35S:CaSLP:GFP, and TRV2:00/35S:GFP plants. D Fresh weight in TRV2:CaNAC035/35S:CaSLP:GFP, TRV2:00/35S:CaSLP:GFP, and TRV2:00/35S:GFP plants before and after 48 h of drought treatment. E Survival rates of TRV2:CaNAC035/35S:CaSLP:GFP, TRV2:00/35S:CaSLP:GFP, and TRV2:00/35S:GFP plants before and after 48 h of drought treatment. Values are means ± SD (n = 3). Asterisks indicate significant difference (*, P < 0.05; **, P < 0.01)

Fig. 11

In this model, Proposed model for CaSLP in response to drought and Pst.DC3000 tolerance stress through three main mechanisms: (1) By interacting with CaNAC035, CaSLP alleviates water loss by stimulating stomatal closure and reducing stomatal density; (2) Upon exposure to drought and SA stresses, CaSLP mediated drought and Pst.DC3000 resistance stress was cleared by ROS; (3) CaNAC035 interacts with CaSLP, and CaNAC035 acts as a transcriptional activator binds to the promoter of CaPR1 to modulate the drought and Pst.DC3000 resistance