Deficiency of a triterpene pathway results in humidity-sensitive genic male sterility in rice

Abstract

In flowering plants, the pollen coat protects the released male germ cells from desiccation and damage during pollination. However, we know little about the mechanism by which the chemical composition of the pollen coat prevents dehydration of pollen grains. Here we report that deficiency of a grass conserved triterpene synthase, OsOSC12/OsPTS1, in rice leads to failure of pollen coat formation. The mutant plants are male sterile at low relative humidity (RH < 60%), but fully male fertile at high relative humidity (>80%). The lack of three major fatty acids in the pollen coat results in rapid dehydration of pollen grains. We show that applying mixtures of linolenic acid and palmitic acid or stearic acid are able to prevent over-dehydration of mutant pollen grains. We propose that humidity-sensitive genic male sterility (HGMS) could be a desirable trait for hybrid breeding in rice, wheat, maize, and other crops.

Fig. 1

Transcription and expression of OsOSC12. a, b Tissue specificity as revealed by northern and western blotting. c, d Temporal profiles during anther development, as shown by real-time PCR and western blotting. S1 to S11 correspond to the stages of anther development as described. The values are presented as means ± s.e., n = 3. e The localization of OsOSC12 mRNA at S9. Hybridization with sense OsOSC12 transcript provided the negative control. f Immuno-localization of OsOSC12 at S10. Hybridization with blocking buffer was used as the negative control. The negative controls for northern and western blotting experiments were, respectively, 18S and 28S rRNA, and the rice heat shot protein Q69QQ6. E epidermis, Msp microspore, T tapetum. Scale bar, 50 μm

Fig. 2

The absence of OsOSC12 results in male sterility. a OsOSC12 mutants induced by either EMS or sodium azide. Boxes and lines indicate the OsOSC12 exons and introns, respectively. TILLING was directed at exons 6–10 and 16–18 (blue box). Mutated nucleotides are marked in red, and the changed nucleotides and the corresponding amino acids are shown for each mutant. b Northern blot analysis of OsOSC12 transcription. c Western blot analysis of OsOSC12 expression. Negative controls as given in Fig. 1. d Self fertility of field-grown WT and mutant plants (30–39 °C, 40–70% RH), based on panicles. The data are presented as mean ± s.d. n = 15. e Germination (upper panel) and pollen tube elongation (lower panel) of WT and E157 pollen, imaged 1 h after pollination. Scale bar, 100 μm

Fig. 3

Characterization of humidity-sensitive genic male sterility. a Germination of WT and mutant pollen on the stigma. T₂ and T₃ refer to the period between, respectively, the time when the pollen came into contact with the stigma and the onset of pollen dehydration, and from the onset of dehydration and complete dehydration. Mp mature pollen, Ps protrusion, Pt pollen tube, St stigma. b Pollen dehydration at 27–32 °C, 30–60% RH. The ratio between the numbers of dehydrated and total pollen grains is presented in the form of mean ± s.d., n = 3. c Seed set of the WT and mutant plants in different humidity. The data are presented as means ± s.d., n = 15. ***P < 0.001, Student’s t tests. GR growth room. d Spikelets of the WT and mutant E157 plant grown in different humidity. Scale bar, 2 cm

Fig. 4

Identification of pollen coat chemicals. TEM analysis of ultramicrotome (a) and cryo-ultramicrotome (b) sections of the WT and E157 pollen wall at S14. c Proposed mode of pollen coat formation during stages S9 to S12. Ba bacula, Cy cytoplasm, In intine, LTPs lipid transfer proteins, Lo locule, Ne nexine, Pc pollen coat (indicated by red arrows), PE primexine, Pla plasma membrane, Te tectum, U ubisch body, Vac vacuole. d Analysis of pollen coat lipids of the WT and E157 plants. The values indicate means ± s.d., n = 5, **P < 0.01, by Student’s t test. e Pollen dehydration rates when treated with pollen coat extracts and chemicals. E157-hexane: E157 pollen treated by hexane, WT-hexane: wild-type pollen treated by hexane, abbreviates of fraction and chemical names are listed in Supplementary Table 4. Times of pollen (n = 100–150) dehydration are presented in the form of mean ± s.d., n = 3. Scale bar, 0.5 μm

Fig. 5

The OsOSC12 committed triterpene ester pathway. a GC–MS profiles of the triterpene alcohol fraction of anther (S12) extracts from OsOSC12 mutant and WT plants. The arrow indicates the peaks present in the WT and the “silent” mutants, but absent in the loss-of-function mutants. Standard, purified poaceatapetol. EIC 189, extracted ion chromatograms at m/z 189. The arrow indicates the poaceatapetol peak. b GC–MS profiles of the triterpene esters of anther (S12) extraction from OsOSC12 mutant and WT plants. 1: poaceatapetol palmitic acid (16:0) ester; 2: poaceatapetol oleic acid (18:1) ester; 3: poaceatapetol stearic acid (18:0) ester; EIC 189, extracted ion chromatograms at m/z 189. c The OsOSC12 committed triterpene pathway for biosynthesis of poaceatapetol esters

Fig. 6

HGMS-based hybrid seed production. a Seed setting rate and hybrid rate of the crosses of E157 and S4928 plants, with the WT plants. The data are presented as mean ± s.d., n = 15 panicles for seed setting rate, n = 300 seeds for hybrid rate. b The variation in RH and temperature at the field site over the period of anthesis. Data were recorded at 1 h intervals by a detector suspended 10 cm above the canopy. c A simplified phylogeny of plant OSCs, based on their coding sequence. A maximum likelihood tree was constructed assuming the GTR + Γ + I model. The gray triangles indicate groups of OSCs derived from the same lineage. Sequences shown in bold indicate poaceatapetol synthase-like genes. d Northern blotting analysis of transcription in the anthers of four major cereal species. e Western blotting demonstrates the presence of OsOSC12-like proteins in the anthers of other cereal species. f The GC/MS profile of the triterpene alcohol fraction of extracts of barley, maize and wheat anthers. Standard, purified poaceatapetol; EIC 189, extracted ion chromatograms at m/z 189. The arrow indicates the poaceatapetol peak