Functional identification of DNA demethylase gene CaROS1 in pepper (Capsicum annuum L.) involved in salt stress

Abstract

Pepper, which is a widely cultivated important vegetable, is sensitive to salt stress, and the continuous intensification of soil salinization has affected pepper production worldwide. However, genes confer to salt tolerance are rarely been cloned in pepper. Since the REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylase that plays a crucial regulatory role in plants in response to various abiotic stresses, including salt stress. We cloned a ROS1 gene in pepper, named CaROS1 (LOC107843637). Bioinformatic analysis showed that the CaROS1 protein contains the HhH-GPD glycosylase and RRM_DME domains. qRT-PCR analyses showed that the CaROS1 was highly expressed in young and mature fruits of pepper and rapidly induced by salt stress. Functional characterization of the CaROS1 was performed by gene silencing in pepper and overexpressing in tobacco, revealed that the CaROS1 positively regulates salt tolerance ability. More detailly, CaROS1-silenced pepper were more sensitive to salt stress, and their ROS levels, relative conductivity, and malondialdehyde content were significantly higher in leaves than those of the control plants. Besides, CaROS1-overexpressing tobacco plants were more tolerant to salt stress, with a higher relative water content, total chlorophyll content, and antioxidant enzyme activity in leaves compared to those of WT plants during salt stress. These results revealed the CaROS1 dose play a role in salt stress response, providing the theoretical basis for salt tolerance genetic engineering breeding in pepper.

Keywords: CaROS1; overexpression; pepper; salt stress; virus-induced gene silencing.

Figure 1

Basic information for CaROS1 in pepper. (A) Structure of CaROS1 gene. Red rectangles and black lines indicate exons and introns, respectively; blue rectangles indicate the upstream and downstream sequence of CaROS1. (B) Physicochemical properties of CaROS1. (C) The predicted secondary structure of CaROS1.

Figure 2

Evolutionary tree and multiple sequence alignment of ROS1 proteins. (A) Evolutionary tree analysis of ROS1 proteins. Red font represents the ROS1 protein in pepper. A yellow background indicates Solanaceae plants, a purple background indicates other plants, and a green background indicates Brassicaceae plants. Si (Solanum lycopersicum L.); Sd (Solanum dulcamara L.); St (Solanum tuberosum L.); Lf (Lycium ferocissimum); Nt (Nicotiana tabacum L.); It (Ipomoea triloba); Pd (Prunus domestica L.); Lj (Lonicera japonica); Ns (Nyssa sinensis); Vd (Vaccinium darrowii); Cr (Catharanthus roseus); Ha (Helianthus annuus L.); At (Arabidopsis thaliana); Cs (Camelina sativa L.); Es (Eutrema salsugineum); Sa (Sinapis alba L.). (B) Alignment of the ROS1 sequences of the Brassicaceae and Solanaceae families. Green lines represent the HhH-GPD domain, and purple lines represent the RRM_DME domain.

Figure 3

Subcellular localization and expression analysis of CaROS1. (A) The 35S::GFP and 35S::CaROS1 plasmids were separately transformed into tobacco leaves for subcellular localization analysis. GFP is shown in green, and nuclear markers in red. Scale bar = 20 μm. (B) Analysis of the expression patterns of CaROS1 in pepper; root (R), stem (S), leaf (L), flower (F), young fruit (YF), and mature fruit (MF). The expression level of CaROS1 in the root was set to 1. (C) Leaves were treated with different concentrations of NaCl (0, 50, 100, and 200 mM) for 20 days, and then the expression pattern of CaROS1 under salt stress was determined using qRT-PCR. “**” indicates P < 0.01.

Figure 4

Phenotypes of TRV: 00 and TRV: CaROS1 plants after 20 days of salt stress. (A) Phenotypes of silenced and control plants after treatment with the indicated concentrations of salt. (B) Nitroblue tetrazolium staining of TRV: 00 and TRV: CaROS1 leaves after salt stress.

Figure 5

Effect of CaROS1 silencing on salt tolerance in pepper. (A) Relative electrolyte conductivity, (B) Proline content (C) MDA content, (D) POD activity, (E) CAT activity (F) SOD activity. “*” indicates P < 0.05 between WT plants and CaROS1-silenced plants; “**” indicates P < 0.01.

Figure 6

Effect of CaROS1 overexpression on salinity stress tolerance in tobacco. (A) Phenotype of WT and OE lines after salt stress, (B) Na⁺ content, (C) relative water content, (D) relative electrolyte conductivity, (E) total chlorophyll content, (F) POD activity, and (G) SOD activity. “*” indicates P < 0.05 between WT and transgenic tobacco; “**” indicates P < 0.01.