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Rice pollen aperture formation is regulated by the interplay between OsINP1 and OsDAF1

Abstract

The aperture on the pollen surface provides an exit for the emerging pollen tube. Apertures exhibit huge morphological variation across plant species—grasses, including rice, possess a complex aperture consisting of an annulus and an operculum—but little is known about how this species-specific cell-surface pattern forms. Here, we report a lectin receptor-like kinase in Oryza sativa, OsDAF1, which is essential for annulus formation and thus for fertility. OsDAF1 is evenly distributed in early microsporocytes but localizes to the distal pre-aperture site at the tetrad stage. We further reveal that the rice orthologue of a key aperture factor in Arabidopsis, OsINP1, has conserved and diversified roles in rice aperture formation. Disruption of OsINP1 prevents formation of the aperture, precluding pollen-tube germination. Furthermore, our results demonstrate that OsINP1 is required for polarization of OsDAF1 via direct protein interaction, suggesting that OsINP1 has an additional role in the formation of annulus that is absent in Arabidopsis. Our study reveals the importance of the aperture for rice grain yield and reveals mechanisms controlling pollen aperture development in cereal species.

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Fig. 1: OsDAF1 is required for rice aperture patterning.
Fig. 2: OsDAF1 assembles at future aperture sites.
Fig. 3: osinp1 is epistatic to osdaf1.
Fig. 4: OsINP1 localizes to future aperture sites.
Fig. 5: The C terminus of OsDAF1 interacts with OsINP1.
Fig. 6: Interaction with OsINP1 is essential for OsDAF1 localization.

Data availability

Source Data for Fig. 5 and Extended Data Fig. 10 are provided with the paper. Sequence data from this article can be found in the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu) under accession numbers LOC_Os02g26160 (OsDAF1) and LOC_Os02g44250 (OsINP1). The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by grants from the National Key Research and Development Program of China (2016YFD0100902), the National Transgenic Major Program Grant (2016ZX08009-003), National Natural Science Foundation of China (31670309), the Innovative Research Team, Ministry of Education, and 111 Project (Grant B14016).

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Authors

Contributions

W.L. and D.Z. designed the project. X.Z. and G.Z. performed most of the experiments. H.Y. performed the map-based cloning. Q.T. performed the rice transformation. L.Z. and Q.T. performed the TEM sections. W.L., X.Z. and N.B. wrote the paper.

Corresponding author

Correspondence to Wanqi Liang.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Plants thanks Anna Dobritsa, Dazhong Zhao and the other, anonymous, reviewers for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Phenotypes and complementation of osdaf1 mutants.

a–c, Comparisons of wild type (WT) and osdaf1-1 mature plants (a), flowering panicles (b) and spikelets (c). le, lemma; pa; palea; gl, glume. d, e, Comparisons of WT, osdaf1-1, osdaf1-2, osdaf1-3 and gOsDAF1 osdaf1-1 flowers (d) and mature panicles (e). f, The proportion of viable pollen produced by wild-type and mutant plants displayed as box plots, showing the first and third quartiles, split by the median and extended to minimum and maximum values. For each line, 50 independent replicates, each of at least 100 pollen grains, were counted. The phenotypes in ae were observed at least three times independently with similar results. Scale bars, 10 cm (a), 1 cm (b, e) and 1 mm (c, d).

Extended Data Fig. 2 Anther transverse sections of wild-type and osdaf1 plants.

Transverse sections of wild-type, osdaf1-1 and osdaf1-2 anthers at different developmental stages. Phenotypes were observed three times independently with similar results. Scale bars, 80 µm.

Extended Data Fig. 3 Pollen germination assay of wild-type and osdaf1 plants.

In vivo (a) and in vitro (b) pollen germination assays of wild-type and osdaf1-1 pollen grains. Arrowheads in a indicate the ovule; arrows in b indicate mutant pollen grains that failed to germinate. These experiments were repeated three times independently with similar results. Scale bars, 200 µm (a) and 50 µm (b).

Extended Data Fig. 4 TEM observation of wild-type and osdaf1-1 pollen aperture areas.

Microspores (left panel) and enlarged aperture areas (right panel) from wild-type (upper panel) and osdaf1-1 (lower panel) at stage 8b (a), stage 9 (b), stage 10a (c), stage 10b (d), stage 11 (e), and stage 12 (f). A, annulus; N, nexine; S, sexine; O, operculum; PM, plasma membrane; MSP, microspore; MP, mature pollen; F, fibrillar-granular layer; Z, Zwischenkörper layer; In, intine. Arrows indicate the trilamellated structures in annulus. Arrowheads indicate the missing annulus structure in osdaf1-1 pollen. These phenotypes were observed three times independently with similar results. Scale bars are indicated.

Extended Data Fig. 5 Identification of OsDAF1.

a, Fine mapping of OsDAF1. Positions and numbers of recombinants of each molecular marker are indicated. The OsDAF1 locus is mapped to a 4138 kb region between Y3’ and YH42-5. AP005534 and AP004850 are accession numbers of BAC clones containing the two molecular markers. b, qRT-PCR analysis of OsDAF1. Quantification was normalized to the expression of the internal control ACTIN. n = 3 biologically independent samples of 50 µg anther from each stage. Data are mean ± SD; dots show data distribution.

Extended Data Fig. 6 Subcellular localizations of OsDAF1.

Subcellular localizations of OsDAF1-eYFP protein in tobacco leaf epidermal cells (left panel) and rice protoplasts (right panel). The subcellular localizations were observed three times independently with similar results. Scale bars are indicated.

Extended Data Fig. 7 Phenotypes of osinp1 mutants.

a, Comparisons of WT, osinp1-1, osinp1-2 and osinp1 osdaf1 flowers (a) and mature panicles (b). These phenotypes were observed at least three times independently with similar results. Scale bars, 1 mm (a) and 1 cm (b).

Extended Data Fig. 8 Subcellular localization of OsINP1.

Subcellular localization of OsINP1-eGFP in tobacco leaf epidermal cells. This subcellular localization was observed three times independently with similar results. Scale bars are indicated.

Extended Data Fig. 9 OsINP1 modifies plasma membrane at aperture sites.

Confocal images of tetrads stained by Calcofluor White (blue, callose wall) and CellMask Deep Red (red, membranous structures) from WT (a), osdaf1 (b), osinp1 (c) and OsINP1-eYFP transgenic plants (d). Merged fluorescent signal from YFP (yellow), Calcofluor White and CellMask Deep Red were shown in d. Arrows indicate the protruded membrane regions. These observations were repeated at least three times independently with similar results. Scale bars, 5 μm.

Extended Data Fig. 10 Signal peptide of OsDAF1 may be essential for protein stability.

a, Expression of full length OsDAF1 (upper panel), 5 amino acid truncated OsDAF1-GFP (middle panel, described in Fig. 5a) or signal peptide truncated OsDAF1-eYFP (lower panel) in tobacco leaf epidermal cells. b, The protein level of full length or truncated OsDAF1 in indicated samples, as determined by western blot. “Empty” represents a tobacco sample without Agrobacterium infiltration. Observations and data were repeated three times independently with similar results. Scale bars are indicated.

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Supplementary Figs. 1 and 2, and Supplementary Table 1.

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Source Data Fig. 5

Unprocessed western blots.

Source Data Extended Data Fig. 10

Unprocessed western blots.

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Zhang, X., Zhao, G., Tan, Q. et al. Rice pollen aperture formation is regulated by the interplay between OsINP1 and OsDAF1. Nat. Plants 6, 394–403 (2020). https://doi.org/10.1038/s41477-020-0630-6

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