Loss of function at RAE2, a previously unidentified EPFL, is required for awnlessness in cultivated Asian rice

Abstract

Domestication of crops based on artificial selection has contributed numerous beneficial traits for agriculture. Wild characteristics such as red pericarp and seed shattering were lost in both Asian (Oryza sativa) and African (Oryza glaberrima) cultivated rice species as a result of human selection on common genes. Awnedness, in contrast, is a trait that has been lost in both cultivated species due to selection on different sets of genes. In a previous report, we revealed that at least three loci regulate awn development in rice; however, the molecular mechanism underlying awnlessness remains unknown. Here we isolate and characterize a previously unidentified EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family member named REGULATOR OF AWN ELONGATION 2 (RAE2) and identify one of its requisite processing enzymes, SUBTILISIN-LIKE PROTEASE 1 (SLP1). The RAE2 precursor is specifically cleaved by SLP1 in the rice spikelet, where the mature RAE2 peptide subsequently induces awn elongation. Analysis of RAE2 sequence diversity identified a highly variable GC-rich region harboring multiple independent mutations underlying protein-length variation that disrupt the function of the RAE2 protein and condition the awnless phenotype in Asian rice. Cultivated African rice, on the other hand, retained the functional RAE2 allele despite its awnless phenotype. Our findings illuminate the molecular function of RAE2 in awn development and shed light on the independent domestication histories of Asian and African cultivated rice.

Keywords: awn; convergent evolution; parallel domestication; rice; signal peptide.

Fig. 1.

Identification and functional characterization of RAE2. (A) The awn is a spine-like extension of the rice lemma. (Left) Awned rice seed anatomy. (Right) Panicles of the awned chromosome segment substitution line GLSL25. The red arrowhead points to the awn. (B–D) Seed phenotypes and graphical genotypes of Koshihikari (O. sativa ssp. japonica; yellow) (B), IRGC104038 (O. glaberrima; blue) (C), and GLSL25 (D). (E–G) Evaluation of transgenic plants with plasmid vector pGWB501 (vector control; V.C.) and RAE2 gene (pRAE2::RAE2): seed phenotype (E), frequency of awned seeds per panicle (F), and awn length (G). No visible awn was observed in pGWB501 (V.C.), indicated as “n.d.” (not detected). (H–J) Evaluation of vector control [pANDA (V.C.)] and RNAi line (RAE2-RNAi): seed phenotype (H), frequency of awned seeds per panicle (I), and awn length (J). (K) rae2/OsEPFL1 amino acid structure of Koshihikari (O. sativa ssp. japonica) and RAE2 of IRGC104038 (O. glaberrima). Yellow triangles indicate insertions. Each colored box represents a peptide region. ma, mature peptide (gray or red); pro, propeptide (green); sp, signal peptide (blue). Red bars indicate cysteine (C) residues. (Scale bars, 1 cm.) The statistical significance was at *P < 0.05 based on a two-tailed Student’s t test. Error bars represent SD of the mean.

Fig. 2.

RAE2 expression pattern and correlation with awn development. (A) Quantitative RT-PCR showing RAE2 mRNA levels in different organs of Koshihikari and GLSL25 (IN, internode; LB, leaf blade; LS, leaf sheath; RO, root; PA, young panicle). OsUBI was used as an internal control. Error bars represent SD of the mean. (B–I) SEM images of spikelets at different developmental stages in Koshihikari (B–E) and GLSL25 (F–I). Developmental stages are classified into Sp7 (B and F), Sp8 (C and G), and post Sp8 (D, E, H, and I) according to Oryzabase classification (www.shigen.nig.ac.jp/rice/oryzabase/devstageineachorgan/list). (Scale bars, 50 µm.) Red arrowheads point to the awn. (J–S) In situ hybridization using antisense probes of rae2 (J–M), RAE2 (O–R), and sense probes (N and S) during spikelet development in Koshihikari and GLSL25. Blue arrowheads indicate the tips of paleae; red arrowheads indicate the tips of lemmas; green asterisks show anthers (an, anther; le, lemma; pa, palea). [Scale bars, 50 µm (J–L and O–Q) and 100 µm (M, N, R, and S).]

Fig. 3.

Distribution of RAE2 protein variants across diverse rice accessions. (A) Awned phenotypes of overexpression lines carrying different RAE2 types (4C, 5C, 6C, 7C). pCAMBIA1380 was used as the vector control. (Scale bar, 1 cm.) (B) Distribution of the four RAE2 protein variants within O. barthii (i; n = 11), O. glaberrima (ii; n = 12), O. rufipogon/O. nivara (iii; n = 65), and O. sativa (iv; n = 42). (C) Geographical distribution of RAE2 protein variants found across these same 130 accession [107 Asian rice accessions (circles) and 23 African rice accession (triangles)]. Symbol sizes are proportional to the number of accessions and are indicated by the numbers in the rectangle. Colors represent each protein variant: yellow, 7C/long; green, 6C/medium; blue, 5C/medium; and red, 4C/short. (D) Awned phenotypes across the four RAE2 protein variants. Numbers of awned (gray bars) and awnless (white bars) accessions for O. barthii (i), O. glaberrima (ii), O. rufipogon/O. nivara (iii), and O. sativa (iv). (E, Upper) Nucleotide diversity of O. sativa individuals (n = 67) relative to nucleotide diversity of O. rufipogon/O. nivara individuals (n = 65) across chromosome 8. The gray box represents reducing relative diversity surrounding RAE2 (green line), consistent with a selective sweep. (E, Lower) Genetic distance between O. sativa and O. rufipogon/O. nivara for all RAE2 types (blue) and dysfunctional ones (red; 4C and 7C). Decreased distance in the dysfunctional class relative to the distance in the “all” class in a 1.5-Mb region surrounding RAE2 (gray box).

Fig. 4.

RAE2 maturation caused by cleavage with SLP1 protease at the spikelet. (A) Immunoblot analysis of RAE2-3×FLAG in transgenic plants with an anti-FLAG antibody. The gray line indicates erased space between the callus and stem lane, although all samples were applied on the same membrane. (B) The expected size of the RAE2-3×FLAG peptide after cleavage in transgenic plants of overexpression constructs: signal peptide (blue), propeptide (green), and mature peptide (pink). (C) In vitro processing assay of recombinant RAE2 peptide incubated with plant extracts of Koshihikari or buffer (mock). The ∼30-kD band is a tag-fused recombinant RAE2 propeptide (indicated by −tag+pro). Asterisks indicate nonspecific bands; the red arrowhead indicates the expected mature RAE2-3×FLAG peptide (∼11 kDa; indicated by −ma). (D) MS ion spectrum for the synthetic peptide (52-AGEEEKVRLGSSPPSCYSK-70), which was cleaved at the position between P65 and S66, indicated by the red line. The full length of the synthetic peptide was cleaved on the other site (+, 54-EEEKVRLGSSPPSCYSK-70; ++, 54-EEEKVRLGSSPP-65). (E) In vitro processing assay of a series of alanine-substituted recombinant RAE2 peptides (mu-RAE2) using the spikelet extract of Koshihikari or buffer (mock). The table (Lower) shows the amino acid sequence around the predicted cleavage point. Other descriptions are the same as in C. (F) Predicted sequence of RAE2, which encodes a 125-amino acid peptide composed of a signal peptide (sp; blue), a propeptide (pro; green), and a mature peptide (ma; pink). The dotted and solid arrows indicate the cleavage sites of Stomagen and EPF2, respectively. See also SI Appendix, Figs. S10 and S12.