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A species-specific functional module controls formation of pollen apertures

Nature Plants volume 7, pages 966–978 (2021)Cite this article

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Abstract

Pollen apertures are an interesting model for the formation of specialized plasma-membrane domains. The plant-specific protein INP1 serves as a key aperture factor in such distantly related species as Arabidopsis, rice and maize. Although INP1 orthologues probably play similar roles throughout flowering plants, they show substantial sequence divergence and often cannot substitute for each other, suggesting that INP1 might require species-specific partners. Here, we present a new aperture factor, INP2, which satisfies the criteria for being a species-specific partner for INP1. Both INP proteins display similar structural features, including the plant-specific DOG1 domain, similar patterns of expression and mutant phenotypes, as well as signs of co-evolution. These proteins interact with each other in a species-specific manner and can restore apertures in a heterologous system when both are expressed but not when expressed individually. Our findings suggest that the INP proteins form a species-specific functional module that underlies formation of pollen apertures.

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Fig. 1: INP2 is a new factor essential for the formation of pollen apertures.
Fig. 2: INP2 is expressed in the male reproductive lineage at the time of aperture formation.
Fig. 3: INP2 is required for INP1 and D6PKL3 accumulation at the aperture domains and both inp1 and inp2 are epistatic to d6pkl3.
Fig. 4: INP1 and INP2 physically interact.
Fig. 5: INP1 and INP2 exhibit similar trends of evolutionary sequence divergence.
Fig. 6: INP1 and INP2 interact in a species-specific manner.
Fig. 7: Tomato orthologues of INP1 and INP2 fail to function in Arabidopsis when expressed individually but gain this ability when co-expressed.
Fig. 8: Certain regions of INP2 mediate its species specificity.

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Data availability

All data supporting the findings of this study are available within the article, Supplementary Information files or from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

Funding for this project was provided to A.A.D. by the US National Science Foundation (MCB-1817835). We acknowledge the support from the US National Institutes of Health grant no. R35GM131760 (to I.B.Z.), the TRONDBUSS program (OSU and Norwegian Science and Technology University) (to I. M.M.), Herta Camerer Gross Postdoctoral Research Fellowship (to R.W.), University graduate fellowships (to P.C. and K.A.), OSU Mayers Undergraduate Summer Research Scholarship (to P.A.), NSF-REU supplement mechanism (to P.A. and A.H.), iCAPS internship from the Center for Applied Science (to A.H.), Undergraduate Research Scholarship from the OSU Arts and Sciences Honors Committee (to A.H.) and a Dr. Elizabeth Wagner Scholarship from the Department of Molecular Genetics at OSU (to A.H.). We thank members of the Dobritsa laboratory for discussions, Y. Zhou for help with phylogenetic analysis, V. Edwards, N. Weyrick and S. Knapp for technical help, D. Somers and D. Mackey for vectors, Arabidopsis Biological Resource Center for DNA stocks and the NCI-subsidized Genomics Facility at the OSU Comprehensive Cancer Center (CCSG:P30CA016058) for sequencing.

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Author notes

  1. These authors contributed equally: Byung Ha Lee, Rui Wang.

Authors and Affiliations

  1. Department of Molecular Genetics and Center for Applied Plant Sciences, Ohio State University, Columbus, OH, USA

    Byung Ha Lee, Rui Wang, Ingrid M. Moberg, Sarah H. Reeder, Prativa Amom, Michelle H. Tan, Katelyn Amstutz, Pallavi Chandna, Adam Helton & Anna A. Dobritsa

  2. Norwegian Science and Technology University, Ålesund, Norway

    Ingrid M. Moberg

  3. Department of Microbiology, Ohio State University, Columbus, OH, USA

    Ekaterina P. Andrianova & Igor B. Zhulin

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Contributions

B.H.L., R.W. and A.A.D. conceived and designed the experiments. B.H.L., R.W., I.M.M., S.H.R., P.A., M.H.T., K.A., P.C., A.H. and A.A.D. performed the experiments. E.P.A., A.A.D. and I.B.Z. performed phylogenetic analysis. B.H.L., R.W., I.M.M., K.A., P.C., E.P.A. and A.A.D. analysed the data. A.A.D. wrote the article and all authors revised and approved the final manuscript.

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Correspondence to Anna A. Dobritsa.

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Peer review information Nature Plants thanks Sheila McCormick, Dabing Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 INP2 and INP1 display similar expression patterns, with both genes showing highest expression in young developing buds.

The RNA-seq data for INP2 (a) and INP1 (b) are from the dataset of Klepikova et al.15 and visualized with the BAR eFP Browser.

Extended Data Fig. 2 INP1 and INP2 proteins both contain the DOG1 domain and have similar structural organization predicted for their C-terminal parts.

a, Protein alignment between INP1 and INP2 proteins. Identical and similar (V/I/L, D/E, K/R, N/Q and S/T) residues are shaded, respectively, in blue and green. The positions of the DOG1 domains predicted by Pfam are indicated by purple lines. b-c, Protein structures predicted by Phyre2 for C-terminal parts of INP1 (b) and INP2 (c) (confidence: >97% for both proteins). In both cases, the modelled regions cover 114 amino acids, which constitute, respectively, 42% of INP1 and 37% of INP2. The same template (c4clvB, nickel-cobalt-cadmium resistance protein NccX from Cupriavidus metallidurans 31a) was selected by the program in both cases.

Extended Data Fig. 3 Alignment of INP2 proteins from representatives of different angiosperm taxa.

The following species were used (from top to bottom): Papaver somniferum (basal eudicots, Papaveraceae), Arabidopsis thaliana (rosids, Brassicaceae) Capsella rubella (rosids, Brassicaceae), Olea europea (asterids, Oleaceae), Mimulus guttatus (asterids, Phrymacae), Manihot esculenta (rosids, Euphorbiaceae), Solanum lycopersicum (asterids, Solanaceae), Nymphaea colorata (basal angiosperms, ANA, Nympheaceae), Oryza sativa (monocots, Poaceae), Zea mays (monocots, Poaceae), Elaeis guineensis (monocots, Arecaceae), Ananas comosus (monocots, Bromeliaceae). The seven regions selected for creating AtINP2/SlINP2 chimeras are indicated by differently coloured rectangles. Aspartate (D) and glutamate (E) residues in the acidic region are shaded in blue. Black shading indicates identical amino acids and grey shading indicates similar amino acids present at the same position in at least half of the aligned proteins.

Extended Data Fig. 4 Arabidopsis INP2 likely contains a transmembrane domain at its N terminus.

Multiple TM discovery algorithms predict existence of the transmembrane domain at the N terminus of INP2 from Arabidopsis thaliana (AtINP2), with the consensus score of 0.85 generated by the plant membrane protein database Aramemnon (AramTMCon).

Extended Data Fig. 5 None of the seven AtINP2 regions is sufficient on its own to convert SlINP2 into a protein able to function in Arabidopsis.

Confocal images of pollen grains produced by the transgenic inp2 plants expressing seven versions of chimeric SlINP2 constructs in which one region at a time was replaced with the corresponding regions from AtINP2. At least 10 independent T1 lines were tested for each construct (≥ 50 pollen grains per line), with similar results. Scale bars = 10 µm.

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Lee, B.H., Wang, R., Moberg, I.M. et al. A species-specific functional module controls formation of pollen apertures. Nat. Plants 7, 966–978 (2021). https://doi.org/10.1038/s41477-021-00951-9

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