OsACOS12, an orthologue of Arabidopsis acyl-CoA synthetase5, plays an important role in pollen exine formation and anther development in rice

Abstract

Background: Sporopollenin is a major component of the pollen exine pattern. In Arabidopsis, acyl-CoA synthetase5 (ACOS5) is involved in sporopollenin precursor biosynthesis. In this study, we identified its orthologue, OsACOS12, in rice (Oryza sativa) and compared the functional conservation of ACOS in rice to Arabidopsis.

Results: Sequence analysis showed that OsACOS12 shares 63.9 % amino acid sequence identity with ACOS5. The osacos12 mutation caused by a pre-mature stop codon in LOC_Os04g24530 exhibits defective sexine resulting in a male sterile phenotype in rice. In situ hybridization shows that OsACOS12 is expressed in tapetal cells and microspores at the transcript level. The localization of OsACOS12-GFP demonstrated that OsACOS12 protein is accumulated in tapetal cells and anther locules. OsACOS12 driven by the ACOS5 promoter could partially restore the male fertility of the acos5 mutant in Arabidopsis.

Conclusions: OsACOS12 is an orthologue of ACOS5 that is essential for sporopollenin synthesis in rice. ACOS5 and OsACOS12 are conserved for pollen wall formation in monocot and dicot species.

Keywords: Anther cuticle; Male sterility; Oryza sativa; OsACOS12; Pollen exine.

Fig. 1

OsACOS12 in O. sativa is an orthologue of ACOS5 in Arabidopsis. a Amino acid sequences alignment of OsACOS12 and ACOS5. The sequences were aligned using Clustal W and displayed using BOXSHADE. b A neighbour-joining phylogenetic tree of OsACOS12 and its orthologues in different species. Bootstrap values are the percentage of 1,000 replicates. The conserved AMP binding domain and fatty acid binding domain are indicated

Fig. 2

Isolation of the rice osacos12 mutant with complete male sterility. a The gene structure and position of the nucleotide change in osacos12. The black boxes indicate exons. b-d The wild-type (WT) plant, osacos12 mutant and complementation plant after the heading stage. e Comparison of the WT plant (left) and osacos12 mutant panicles (right) at the heading stage. f The spikelets of the WT plant (left) and an osacos12 mutant (right) after removing the palea. g The anther of a WT plant (left) and an osacos12 mutant (right). h Alexander staining of the WT plant (left) and an osacos12 mutant anther (right). i and j Identification of the OsACOS12 gene in a WT plant and osacos12 mutant by sequencing (position 1000). Bars = 100 μm in e-g and 200 μm in h

Fig. 3

The defective anther cuticle and pollen sexine formation in osacos12. a and b SEM images for the WT and osacos12 anthers. c-j SEM observation for the epidermal surface of the WT (c) and osacos12 (g) anthers, the inner surface of WT (d) and osacos12 (h) anthers, and the pollen grains in WT (e and f) and osacos12 (i and j) anthers. Or, orbicule; Bars = 500 μm in a and b, 100 μm in e, i, 10 μm in c, g, f, j and 5 μm in h, d. k-t Semi-thin cross-sectional analysis of anther development of WT (k-o) and the osacos12 mutant (p-t) during the anther development stages. E, epidermis; En, endothecium; ML, middle layer; T, tapetum; MMC, microspore mother cell; Tds, tetrads; Msp, microspore. Bars = 20 μm. u-z TEM observation for WT (u-w) and osacos12 (x-z) pollen development from stages 8–10. The boxed image on the right of each panel was enlarged from the left region. AEX, abnormal exine; Ba, bacula; E, epidermis; En, endothecium; Ex, exine; Msp, microspore; Ne, nexine, PE, primexine; Se, sexine; T, tapetum; Tds, tetrads. Bars = 5 μm and 500 nm in u, x, 2 μm and 500 nm in v, y, 5 μm and 1 μm in w, 2 μm and 1 μm in z

Fig. 4

Anther cuticle wax constitutions in WT and osacos12. a The total amount of anther wax per unit of anther surface area. b The amounts of anther wax per unit of anther surface area. Compound names are abbreviated as follows: C14, myristic acid; C18, stearic acid; C18:3, linolenic acid; C20, arachidic acid; C24, lignoceric acid; C26, hexacosanoic acid; C26, hexacosane; C27, heptacosane; C30, triacontane; C32, dotriacontane; C33, tricosane; C35, pentatriacontane; C36, hexatriacontane; C27, 1-heptacosanol; C28, 24-epicampesterol; C29, sitosterol; C30, 1-triacontanol. Values are the mean ± SD (n = 3). *, P < 0.05; **, P < 0.01 (Student’s t test)

Fig. 5

OsACOS12 is specifically expressed in the anther. a RT-PCR analysis of RNA isolated from various tissues using OsACOS12 and OsACTIN primer sets. Le, lemma; Pa, palea; L2.5, Glumes length 2.5 mm; L3.0, Glumes length 3.0 mm; L3.5, Glumes length 3.5 mm; L4.0, Glumes length 4.0 mm; L5.6, Glumes length 5.6 mm. b Quantitative real-time PCR analysis of OsACOS12. The OsACTIN gene served as the reference. Data are shown as the mean ± SD (n = 3). c-j In situ hybridization of OsACOS12 in WT anthers. The anthers at the MMC stage (c), early meiosis stage (d), tetrad stage (e and f), microspore release stage (g), and microspore vacuolate stage (h) hybridized with an OsACOS12 antisense probe. The anthers at the tetrad stage (i-j) hybridized with an OsACOS12 sense probe. Msp, microspore; T, tapetum; Tds, tetrads. MMC, microspore mother cell; MC, meiotic cell; Dy, dyad cell. Bars = 50 μm. k-p Fluorescence confocal images of the OsACOS12-GFP fusion proteins at different stages. The green channel shows the GFP expression (530 nm), and the red channel shows the chlorophyll autofluorescence (>560 nm). The bright-field images of (p) show that these fusion proteins are not localized to the microspores. Bars = 10 μm; 100 μm

Fig. 6

The OsACOS12 could partially restore acos5 fertility. a and b Structural representation of the proACOS5:OsACOS12 and the proOsACOS12:OsACOS12 constructs. c-f The main stem of the Col (c), acos5 (d), proACOS5:OsACOS12 with a acos5/acos5 background (e), and proOsACOS12:OsACOS12 with a acos5/acos5 background (f) plants. g-j Alexander staining of the anthers from the Col (g), acos5 (h), proACOS5:OsACOS12 (i), and proOsACOS12:OsACOS12 transgenic lines (j). Bars = 100 μm. k-n SEM examination of the dehiscent pollen grains of the wild type (k), acos5 (l), proACOS5:OsACOS12 (m), and proOsACOS12:OsACOS12 transgenic lines (n). Bars = 5 μm. o RT-PCR analysis of ACOS5 or OSACOS12 expression in the flower buds of the Col, acos5 plants and the proACOS5:OsACOS12 and proOsACOS12:OsACOS12 transgenic lines. TUBULIN was used to monitor the cDNA yield and integrity of the samples