APICAL SPIKELET ABORTION (ASA) Controls Apical Panicle Development in Rice by Regulating Salicylic Acid Biosynthesis

Abstract

Panicle degradation causes severe yield reduction in rice. There are two main types of panicle degradation: apical spikelet abortion and basal degeneration. In this study, we isolated and characterized the apical panicle abortion mutant apical spikelet abortion (asa), which exhibits degeneration and defects in the apical spikelets. This mutant had a pleiotropic phenotype, characterized by reduced plant height, increased tiller number, and decreased pollen fertility. Map-based cloning revealed that OsASA encodes a boric acid channel protein that showed the highest expression in the inflorescence, peduncle, and anther. RNA-seq analysis of the asa mutant vs wild-type (WT) plants revealed that biological processes related to reactive oxygen species (ROS) homeostasis and salicylic acid (SA) metabolism were significantly affected. Furthermore, the asa mutants had an increased SA level and H₂O₂ accumulation in the young panicles compared to the WT plants. Moreover, the SA level and the expression of OsPAL3, OsPAL4, and OsPAL6 genes (related to SA biosynthesis) were significantly increased under boron-deficient conditions in the asa mutant and in OsASA-knockout plants. Collectively, these results suggest that the boron distribution maintained by OsASA is required for normal panicle development in a process that involves modulating ROS homeostasis and SA biosynthesis.

Keywords: Oryza sativa; anther; apical panicle abortion; reactive oxygen species; salicylic acid.

FIGURE 1

Phenotypic characterization of the asa mutant. (A) Phenotypic comparison between mature wild-type (WT) and asa mutant. Immature panicles phenotypic comparison between WT (B) and asa mutant (C). (D) Mature panicles phenotypic comparison between WT and asa mutant. (E) Statistics of seed setting rate of WT and asa mutant. PB, primary branch; SB, secondary branch. Comparison of plant height (F), panicle length (G), panicle number (H), length of flag leaf (I), and width of flag leaf (J) between WT, hybrid type (asa/ASA), and asa mutant. Values are mean ± SD (n = 12); asterisks indicate significant differences (*P < 0.05, **P < 0.01) according to the Student’s t-test compared with the WT.

FIGURE 2

Spikelet morphology of wild-type (WT) and asa mutant. (A–E) Spikelet morphology of WT and asa mutant. The WT (A), different phenotype types of asa mutants (B–E). Bars = 1 mm. (F–J) Anther and pistil phenotype of WT and asa mutants. The WT (F), different phenotype types of asa mutants (G–J). Bars = 1 mm. Stigma morphology of WT (K) and asa mutant (L). Bars = 1 mm. (M,N) Spikelet meristems at spikelet specification developmental stage. The WT (M) and asa mutant (N). Stars mark the stamen primordia. Bars = 50 μm. (O,P) Spikelet meristems at stamen and pistil primordia differentiation stage. The WT (O) and asa mutant (P). Bars = 200 μm. (Q,R) Epidermal surface of spikelets. The WT (Q), asa mutant (R). Bars = 500 μm. Potassium iodide staining of mature pollen grains of apical spikelets in WT (S) and asa mutant (T). Bars = 100 μm. Scanning electron microscopy (SEM) observation of mature pollen grains of apical spikelets in WT (U) and asa mutant (V). Bars = 100 μm. le, lemma; pa, palea; pi, pistil; st, stamen; lo, lodicule; glo, glume-like organ; asl, abnormal sterile lemmas.

FIGURE 3

Transverse section and comparison between wild-type (WT) and asa anthers. Semithin sections of WT (A,C) and asa (B,D) anthers at middle young microspore stage, and mature pollen stage. Bars = 50 μm. (E–J) Transmission electron microscopy analysis of WT and asa anthers. Anthers of the WT (E) and asa (H), young microspores in WT (F) and asa (I), tapetal cells of WT (G) and asa (J). Bars = 10 μm in (E); 50 μm in (F,H); and 2 μm in (G,I,J). E, epidermis; En, endothecium; T, tapetum; Msp, microspores; MP, mature pollen; ML, middle layer; ER, endoplasmic reticulum; Lip, lipidosomes.

FIGURE 4

Map-based cloning of asa. (A) Fine mapping of asa. Molecular markers and numbers of recombinants are indicated above and below the bars, respectively. The gray and black boxes indicate untranslated regions and exons, respectively. (B) Alignment of the coding nuclear acid sequence of OsASA and Osasa. (C) Genomic structure of the OsASA gene. Black boxes indicate exons, and the sgRNA target sequence is underlined in blue. (D–F) Genetic confirmation of the OsASA gene. Wild type (D), complemented with OsASA cDNA driven by the UBIQUTIN promoter introduced into asa mutant (OsASA-OE/asa) (E), the knockout plant by CRISPR-Cas9 targets the first exon of OsASA(cr-OsASA) (F).

FIGURE 5

Expression pattern of OsASA. (A) Relative expression of OsASA in various tissues between wild-type and asa mutant. Rice UBIQUITIN gene was used as an internal control. 0–4, 4–8, and 8–12 cm present developing panicles at the 0–4, 4–8, and 8–12 cm stages. Values are mean ± SD. (B) Promoter activity of OsASA by GUS staining at early developmental stages of panicle. Different stages are shown as indicated by panicle length. Bars = 1 mm. (C–G) Promoter activity of OsASA by GUS staining at late developmental stages of panicles. Different stages are shown as indicated by panicle length, 2.5 cm (C), 3.5 cm (D), 5 cm (E), 7.5 cm (F), and 13 cm (G). Bars = 1 cm. (H) Promoter activity of OsASA as shown by GUS staining in spikelets. Bars = 1 mm.

FIGURE 6

Reactive oxygen species accumulation in asa. (A) DAB staining of spikelets in wild-type and asa mutant. Bars = 1 mm. (B) Quantification of H₂O₂ from 3-cm stage panicles of the wild-type and asa mutant. Data are means ± SD (n = 3). Asterisks indicate significant difference from the wild type (**P < 0.01), as determined by Student’s t-test compared with the wild type.

FIGURE 7

Transcriptome and hormone analysis of wild-type and asa mutant. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of differentially expressed genes. (B) Hormone content of 3-cm young panicles in wild-type and asa mutant. SA, SAG, ABA, JA, IAA, and JA-ILE were measured. SA, salicylic acid; SAG, SA glucoside; ABA, abscisic acid; JA, jasmonic acid; IAA, indole acetic; JA-ILE, jasmonic acid-isoleucine. Data are means ± SD (n = 3), asterisks indicate significant difference from the wild type (*P < 0.05, **P < 0.01), as determined by Student’s t-test compared with the wild type. (C–G) RT-PCR analysis of candidate genes in the wild-type and asa mutant. Data are means ± SD (n = 3).

FIGURE 8

asa mutants were sensitive to boron and affect salicylic acid biosynthesis. Growth status of wild-type and asa mutant under 0 μM boron concentration (A), 15 μM boron concentration (B), and 150 μM boron concentration (C). Bars = 5 cm. (D,E) Free salicylic acid and SA glucoside content of wild-type, asa mutant, and CRISPR-OsASA plants. Wild-type, asa, and CRISPR-OsASA plants were grown for 3 weeks in medium containing 15 μM boron (+B) or without boron (-B). Data are means ± SD (n = 3). Asterisks indicate significant difference from the wild type (**P < 0.01), as determined by Student’s t-test compared with the wild type. (F–H) Expression level of OsPAL3, OsPAL4, and OsPAL6 in response to -B treatment. Wild-type, asa, and CRISPR-OsASA plants were grown for 3 weeks in medium containing 15 μM boron (+B) or without boron (-B). Data are means ± SD (n = 3).