OsPEX11, a Peroxisomal Biogenesis Factor 11, Contributes to Salt Stress Tolerance in Oryza sativa

Abstract

Peroxisomes are single membrane-bound organelles, whose basic enzymatic constituents are catalase and H₂O₂-producing flavin oxidases. Previous reports showed that peroxisome is involved in numerous processes including primary and secondary metabolism, plant development and abiotic stress responses. However, knowledge on the function of different peroxisome genes from rice and its regulatory roles in salt and other abiotic stresses is limited. Here, a novel prey protein, OsPEX11 (Os03g0302000), was screened and identified by yeast two-hybrid and GST pull-down assays. Phenotypic analysis of OsPEX11 overexpression seedlings demonstrated that they had better tolerance to salt stress than wild type (WT) and OsPEX11-RNAi seedlings. Compared with WT and OsPEX11-RNAi seedlings, overexpression of OsPEX11 had lower level of lipid peroxidation, Na⁺/K⁺ ratio, higher activities of antioxidant enzymes (SOD, POD, and CAT) and proline accumulation. Furthermore, qPCR data suggested that OsPEX11 acted as a positive regulator of salt tolerance by reinforcing the expression of several well-known rice transporters (OsHKT2;1, OsHKT1;5, OsLti6a, OsLti6b, OsSOS1, OsNHX1, and OsAKT1) involved in Na⁺/K⁺ homeostasis in transgenic plants under salinity. Ultrastructural observations of OsPEX11-RNAi seedlings showed that they were less sensitive to salt stress than WT and overexpression lines. These results provide experimental evidence that OsPEX11 is an important gene implicated in Na⁺ and K⁺ regulation, and plays a critical role in salt stress tolerance by modulating the expression of cation transporters and antioxidant defense. Thus, OsPEX11 could be considered in transgenic breeding for improvement of salt stress tolerance in rice crop.

Keywords: Oryza sativa; OsPEX11; protein interaction; salt tolerance; transgene.

FIGURE 1

OsCYP2 interacts with OsPEX11 protein by in vivo assay. Yeast clones were grown on DDO/Xor QDO/X/A plates that contained X-α-gal. The blue color in the colony co-transformed with BD-OsCYP2 and AD-OsPEX11 indicates interaction between the two proteins. The empty pAD vector was used as negative control. DDO/X medium: SD/-Trp-Leu/X-α-gal; QDO/X/A medium: SD/-Trp-Leu-Ade-His/X-α-gal/AbA; AD-OsPEX11: pGADT7+OsPEX11; BD: pGBKT7 vector; BD-OsCYP2: pGBKT7+OsCYP2.

FIGURE 2

Detection of GST (glutathione-S-transferase) and recombinant GST-OsCYP2. The GST and GST-OsCYP2 proteins were extracted and purified from E. coli strain BL21 using MagneGST Pull Down System (Promega). The proteins were analyzed with 12% SDS-PAGE. (A) SDS-PAGE gel was stained by coomassie brilliant blue R-250. (B) Horseradish Peroxidase DAB staining was used in western blotting. 1: GST-OsCYP2. 2: GST. M: Pre-Stained Protein Marker.

FIGURE 3

Confirmation of interaction between OsCYP2 and OsPEX11 protein using in vitro assay. Recombinant glutathione-S-transferase (GST) and GST-OsCYP2 was used as bait and incubated with 1 μg of prey protein (His-OsPEX11), respectively. After incubation, GST and GST-OsCYP2 were retrieved with glutathione beads, and the pulled-down proteins were detected on Western blots with antibodies to Histidine. GST: pGEX-4T-1; GST-OsCYP2: pGEX-4T-1+OsCYP2; His-PEX11: pET28a+OsPEX11.

FIGURE 4

The morphological changes of 10-day-old wild type (WT), OsPEX11 over-expression (OE1) and RNAi (RNAi1) seedlings under control and salt stress (200 mM NaCl). The seedlings were treated with 200 mM NaCl for 24 h. (A) WT, (B) WT + NaCl, (C) 35S-OsPEX11, (D) 35S-OsPEX11 + NaCl, (E) RNAi-OsPEX11, and (F) RNAi-OsPEX11 + NaCl.

FIGURE 5

Relative transcript level of OsPEX11 responses to H₂O and 200 mM NaCl stress in 10-day-old WT, overexpression (OE1 and OE2), and RNA interference (RNAi1 and RNAi2) lines. The seedlings were treated with 200 mM NaCl for 24 h. Relative expression levels were measured by qRT-PCR analysis using actin as an internal standard. The relative fold expression of CK was considered as 1. Values are means of three biological replicates and significant differences between means, as determined by Turkey test (P < 0.05), are indicated by different letters.

FIGURE 6

Comparison of lipid peroxidation and ROS scavenging in leaves of 10-day-old WT and OsCYP2-transgenic (OE1 and OE2, RNAi1 and RNAi2) seedlings under control and salt stress. The rice seedlings were treated with 200 mM of NaCl for 24 h. The Na⁺/K⁺ ratio (A) and activities of antioxidant enzymes MDA (B), SOD (C), POD (D), CAT (E), and proline content (F) were assayed. Values are means of three biological replicates followed by the same letter did not significantly differ at P ≤ 0.05 according to Turkey’s multiple range test.

FIGURE 7

Relative fold expression of the genes encoding Na⁺ and K⁺ transporter proteins. (A) OsHKT2;1; (B) OsHKT1;5; (C) OsLti6a; (D) OsLti6b; (E) OsSOS1; (F) OsNHX1; (G) OsAKT1 in the leaves of 10-day-old rice seedlings treated with 200 mM of NaCl. The relative fold expression of CK was considered as 1. An actin was used as internal standard. Values are the means of three biological replications ±SD. Variants possessing the same letter are not statistically significant at P < 0.05.

FIGURE 8

Electron micrographs of leaf mesophyll cells of 10-day-old hydroponically grown seedlings of O. sativa under control condition and 200 mM NaCl treatment. Under control condition, grana (G), mitochondria (MTC), and plastoglobuli (PG) well developed. TEM micrographs of leaf mesophyll cells of WT showed that ultrastructure of chloroplast is disrupted with enlarged size PG under 200 mM NaCl treatment (D). Chloroplasts of OsPEX11-OE1 plants maintained normal shape under salt stress condition with dilated thylakoids arrangement (E), whereas, chloroplasts of OsPEX11-RNAi1 plants were round in shape with fewer numbers of PG and swollen mitochondria (MTC) as compared to its respective control (F). (A–C) control: WT (Left); OsPEX11-OE1 (Middle); OsPEX11-RNAi1 (Right); (D–F) 200 mM NaCl: WT (Left); OsPEX11-OE1 (Middle); OsPEX11-RNAi1 (Right).