Molecular Characterization of Rice OsLCB2a1 Gene and Functional Analysis of its Role in Insect Resistance

Abstract

In plants, sphingolipids, such as long-chain bases (LCBs), act as bioactive molecules in stress responses. Until now, it is still not clear if these lipids are involved in biotic stress responses to herbivore. Herein we report that a rice LCB gene, OsLCB2a1 encoding a subunit of serine palmitoyltransferase (SPT), a key enzyme responsible for the de novo biosynthesis of sphingolipids, plays a critical role in plant defense response to the brown planthopper (BPH) attack and that its up-regulation protects plants from herbivore infestation. Transcripts of OsLCB2a1 gene in rice seedlings were increased at 4 h, but decreased at 8-24 h after BPH attack. Sphingolipid measurement profiling revealed that overexpression of OsLCB2a1 in Arabidopsis thaliana increased trihydroxylated LCB phytosphingosine (t18:0) and phytoceramide by 1.7 and 1.3-fold, respectively, compared with that of wild type (WT) plants. Transgenic Arabidopsis plants also showed higher callose and wax deposition in leaves than that of WT. Overexpression of OsLCB2a1 gene in A. thaliana reduced the population size of green peach aphid (Myzus persicae). Moreover, the electrical penetration graph (EPG) results indicated that the aphids encounter resistance factors while reaching for the phloem on the transgenic plants. The defense response genes related to salicylic acid signaling pathway, remained uplgulated in the OsLCB2a1-overexpressing transgenic plants. Our data highlight the key functions of OsLCB2a1 in biotic stress response in plants.

Keywords: Arabidopsis; Myzus persicae; electrical penetration graph; insect resistance; long-chain base; serine palmitoyltransferase; sphingolipids.

Figure 1

(A) Comparison of the pyridoxal 5′-phosphate (PLP)-binding motif of LCB2a. Species are indicated as follows: Nb, Nicotiana benthamiana; At, Arabidopsis thaliana; Os, Oryza sativa; Hs, Homo sapiens; Sc, Saccharomyces cerevisiae. An asterisk marks the PLP-binding lysine residue. (B) Phylogenetic tree of LCB2a homolog in different organism.

Figure 2

Expression of OsLCB2a1 under BPH attack. Two weeks rice seedlings were infested with 3rd instar BPH nymph (20 nymphs/ seedling). Total RNA was isolated from rice seedlings infested by BPH at 0, 1, 2, 4, 8, 24 h and reversely transcribed into cDNA used as template in quantitative RT-PCR analysis. Values are mean ± SE (n = 5). The asterisks indicate statistically significant differences between the treatments (One way ANOVA followed by Tukey's tests, P < 0.05).

Figure 3

Measurement of sphingolipids in WT and OE plants. (A) Total LCBs and ceramides in indicating plants. (B) Free LCBs composition. (C) LCB moiety distribution of ceramide species. Values represent means ± SE from three independent experiments. Different letters indicate significant differences (P < 0.05) between the treatments.

Figure 4

Chromogenic assay of callose in leaves of the transgenic Arabidopsis plant and WT. Leaves of 4 weeks old were stained for 4 h in darkness with aniline blue solution and deposition of callose in leaves was observed under fluorescence microscope of ultraviolet excitation. (A) Control (WT) (B) Transgenic Plant (C) Expression analysis of plant callose-related genes in transgenic (OE) and WT plants. Two callose synthase-encoding genes, GSL1 and GSL5 were analyzed. The AtActin8 gene was used as reference control. The data are the means ± SE from three independent experiments. The asterisks indicate statistically significant differences between the transgenic and control plants (ANOVA followed by Tukey's tests, P < 0.05). Callose deposition were shown by arrows.

Figure 5

Field Emission Scanning Electron Microscopy (FESEM) analysis of cuticle wax depositions. Adaxial side of 4-week-old Arabidopsis rosette leaves of WT and OE were observed under 6000 × magnification. (A) WT showed only little wax deposition on leaf surface. (B) The leaf surfaces of transgenic plants with high wax deposition. Wax crystals were shown by arrows.

Figure 6

Population density of the green peach aphid in WT and OE Arabidopsis plants. Values represent means ± SE from 15 independent experiments. Different letters indicate significant differences (P < 0.05) among the treatments.

Figure 7

(A–F) Content of (nmolg⁻¹FW) different LCBs and Ceramides in Arabidopsis after treated with phytosphingosine. Four-week-old plants were sprayed with phytosphingosine or methanol as control (solvent of phytosphingosine) for 8 h. Values are expressed as the mean ± SE of three independent experiments. Different letters indicate significant differences (P < 0.05) among the treatments (Arabidopsis and phytosph treatments). Sph, sphingosine; Dhsph, dihydrosphingosine; Cer, ceramide; FW, fresh weight.

Figure 8

Population density of the green peach aphid in phyto-sph treated WT and over-expressed Arabidopsis plants. Values represent means ± SE from five independent experiments. Different letters indicate significant differences among the treatments (two-factor ANOVA, P < 0.05).

Figure 9

Expression patterns of plant defense-response genes. PAD4, EDS1, SA synthesis-related genes NPR1 is a key regulator of SA-dependent systemic acquired resistance. LOX2 and VSP2 are the JA synthesis-related genes. EIN2 and EFR1 is the ethylene signaling pathway receptor gene. AtActin8 was used as reference control. In all panels, the mean is based on the average of three independent experiments. One-way ANOVA was used to generate the P-values (^*P < 0.05).