Allene oxide synthase 1 contributes to limiting grain arsenic accumulation and seedling detoxification in rice

Abstract

Arsenic (As) is a cancerogenic metalloid ubiquitously distributed in the environment, which can be easily accumulated in food crops like rice. Jasmonic acid (JA) and its derivatives play critical roles in plant growth and stress response. However, the role of endogenous JA in As accumulation and detoxification is still poorly understood. In this study, we found that JA biosynthesis enzymes Allene Oxide Synthases, OsAOS1 and OsAOS2, regulate As accumulation and As tolerance in rice. Evolutionary bioinformatic analysis indicated that AOS1 and AOS2 have evolved from streptophyte algae (e.g. the basal lineage Klebsormidium flaccidum) - sister clade of land plants. Compared to other two AOSs, OsAOS1 and OsAOS2 were highly expressed in all examined rice tissues and their transcripts were highly induced by As in root and shoot. Loss-of-function of OsAOS1 (osaos1-1) showed elevated As concentration in grains, which was likely attributed to the increased As translocation from root to shoot when the plants were subjected to arsenate [As(V)] but not arsenite [As (III)]. However, the mutation of OsAOS2 (osaos2-1) showed no such effect. Moreover, osaos1-1 and osaos2-1 increased the sensitivity of rice plants to both As(V) and As(III). Disrupted expression of genes involved in As accumulation and detoxification, such as OsPT4, OsNIP3;2, and OsOASTL-A1, was observed in both osaos1-1 and osaos2-1 mutant lines. In addition, a As(V)-induced significant decrease in Reactive Oxygen Species (ROS) production was observed in the root of osaos1-1 but not in osaos2-1. Taken together, our results indicate OsAOS1 modulates both As allocation and detoxification, which could be partially attributed to the altered gene expression profiling and ROS homeostasis in rice while OsAOS2 is important for As tolerance.

Keywords: Arsenic tolerance; Evolutionary bioinformatics; Jasmonic acid; Oryza sativa L.; ROS homeostasis.

Fig. 1

Evolutionary analysis of allene oxide synthase 1 (AOS1) homologues in land plants and algal species. A The phylogenetic tree of AOS1 homologues identified from the representative species from various linkages. The orthologues from Klebsormidium flaccidum, Oryza sativa, Arabidopsis thaliana, Marchantia polymorpha, Physcomitrella patens were highlighted with red asterisks. B Conserved motifs through multiple protein sequences alignment of AOS1 homologues from selected species. C Sequence alignment with the deduced protein fragments containing the mutation site of t of osaos1–1

Fig. 2

Expression pattern of allene oxide synthases (AOS) in rice. A The relative expression of OsAOS1–4 in the organs of rice plants cultured in the paddy field harvested at filling stage. The response of OsAOS1–1 to various condition of As(III) (B) and As(V) (C)

Fig. 3

Yield-related traits and mineral concentrations in the grain of osaos1–1, osaos2–1 and WT. Seed setting rate (A), grain weight (B) and grain yield per plant (C) of osaos1–1, osaos2–1 and WT grown in soil-contained pots until mature. D The concentrations of As, Cu, Fe, Mn and Zn in the brown rice of the mutant lines and WT. Values are means of three independent replicates ± SE (n = 4 for A-C, and n = 10 for D). Different letters indicate significant differences in each graph (p < 0.05)

Fig. 4

As accumulation and distribution in the seedlings of osaos1–1, osaos2–1 and WT at vegetative growth stage. As concentration in the root (A), shoot (B) of the plants harboring 3–4 leaves subjected to 2 or 5 μM As(V) for 6 days. Total As taken up by the plants (C) and the ratio of As translocated from root to shoot of each genotype (D) was calculated. E Increased plant height before and after the As treatments. F As in the xylem sap collected from the 30-day-old plants treated with 2 μM As(V) for 0.5 hour. Values are means of three independent replicates ± SE (n = 4). Different letters indicate significant differences in each graph (p < 0.05)

Fig. 5

Plant growth and biomass of osaos1–1, osaos2–1 and WT under As(V) treatment. Ten-day-old seedlings of the three genotypes subjected to 0 (A, E), 2 (B, F), 5 (C, G) or 10 μM As(V) (D, H) for 10 days. The aboveground tissues (A-D) and whole plants (E-H) were recorded, and the youngest leaves of the plants under 10 μM As(V) were zoomed in a-c. The dry weight of root (I) and shoot (J) of the plants were measured. Values are means of three independent replicates ± SE (n = 4). Different letters indicate significant differences in each graph (p < 0.05)

Fig. 6

Relative root elongation of osaos1–1, osaos2–1 and WT subjected to As. Two-day-old seedlings of the plants were treated with 15 μM As(III) or 2 μM As(V) for 2 days, the length of the seminal root was measured (A) and the relative root elongation rate was calculated (B). Values are means of three independent replicates ± SE (n = 10). Different letters indicate significant differences in each graph (p < 0.05)

Fig. 7

Expression of genes involved in As accumulation and detoxification in rice. After being treated with 2 or 5 μM As(V) for 6 days (Same condition as that in Fig. 4A-D), the root and shoot were harvested for RNA extraction and 1-st strand cDNA synthesis. The relative expression levels of the genes were compared through quantitative real-time PCR. Values are means of three independent replicates ± SE (n = 3). Different letters indicate significant differences in each graph (p < 0.05). Abbreviations: Phosphate (P) transporters (OsPT1,OsPT4, OsPT8), Nodulin 26-like Intrinsic Protein (OsNIP3;2), O-acetylserine (thiol) lyase (OsOASTL-A1)

Fig. 8

ROS accumulation in the roots of osaos1–1, osaos2–1 and WT subjected to As(V). After being treated with 2 μM As(V) for 1 or 6 hours, the seminal root of the 3-day-old seedlings were stained with 10 μM CM-H₂DCFDA for 1 hour and was washed for 3 times was employed for ROS detection. The representative figures were present in (A) and (B). The signal intensity of root tip (0–5 mm) (C) and basal root region (7–12 mm) (D) was calculated from 5 independent replicates. Values are means of three independent replicates ± SE (n = 5). Different letters indicate significant differences in each graph (p < 0.05). Scale bar = 100 μm

Fig. 9

OsAOS1 and OsAOS2 contribute to differential accumulation and detoxification of As in rice. OsAOS1 (black routes) and OsAOS2 (green routes) are the key enzymes for the biosynthesis of JA in rice. OsAOS1 limited As accumulation in rice grain and As detoxification, which is partially attributed to the indirectly altered expression of genes such as OsPT1, OsPT4, OsNIP3;2, OsLsi6 and OsOASTL-A1. ROS homeostasis likely contributes to the regulation of As transport and tolerance mediated by OsAOS1 but not OsAOS2. Increased As sensitivity in the loss-of-function lines of OsAOS2 is largely depended on the changed expression of OsPT8, OsNIP3;2, OsLsi6 and OsOASTL-A1