Abstract
Pollen apertures, the characteristic gaps in pollen wall exine, have emerged as a model for studying the formation of distinct plasma membrane domains. In each species, aperture number, position, and morphology are typically fixed; across species they vary widely. During pollen development, certain plasma membrane domains attract specific proteins and lipids and become protected from exine deposition, developing into apertures. However, how these aperture domains are selected is unknown. Here, we demonstrate that patterns of aperture domains in Arabidopsis are controlled by the members of the ancient ELMOD protein family, which, although important in animals, has not been studied in plants. We show that two members of this family, MACARON (MCR) and ELMOD_A, act upstream of the previously discovered aperture proteins and that their expression levels influence the number of aperture domains that form on the surface of developing pollen grains. We also show that a third ELMOD family member, ELMOD_E, can interfere with MCR and ELMOD_A activities, changing aperture morphology and producing new aperture patterns. Our findings reveal key players controlling early steps in aperture domain formation, identify residues important for their function, and open new avenues for investigating how diversity of aperture patterns in nature is achieved.
Highlights
As part of cell morphogenesis, cells often form distinct plasma-membrane domains that acquire specific combinations of proteins, lipids, and extracellular materials
60 we perform a detailed analysis of this mutant and identify the MCR gene. We demonstrate that it belongs to the ancient family of ELMOD proteins, and that together with another member of this protein family in Arabidopsis, ELMOD_A, MCR acts at the beginning of the aperture formation pathway as a positive regulator of aperture domain specification
We further demonstrate that a third member of this family, ELMOD_E, has an ability to influence the number, positions, and morphology of aperture domains, and we identify specific protein residues critical for this ability
Summary
As part of cell morphogenesis, cells often form distinct plasma-membrane domains that acquire specific combinations of proteins, lipids, and extracellular materials. To confirm that these defects were caused by mutations in ELMOD_A and not off-site CRISPR targeting events, as well as to identify the ELMOD_A regulatory regions, we created two ELMOD_A genomic constructs driven by the 2-kb region upstream of its start codon – EApr:gELMOD_A (which included a 0.3-kb ELMOD_A 3’ UTR) and EApr:gELMOD_A238 YFP (tagged with YFP and lacking the ELMOD_A 3’ UTR) – and transformed them into the mcr elmod_a double mutant, which no longer carried the CRISPR/Cas transgene Both constructs successfully rescued formation of apertures (5/5 and 31/33 T1 plants, respectively, Figure 4H– 4I’), indicating the selected promoter region is sufficient for ELMOD_A functional expression. When both residues undergo MCR-like changes, ELMOD_E loses its neomorphic activity, instead becoming capable of carrying out the MCR role in aperture formation
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