LAP3, a novel plant protein required for pollen development, is essential for proper exine formation

We isolated lap3-1 and lap3-2 mutants in ascreen for pollen that displays abnormal stigma binding.Unlike wild-type pollen, lap3-1 and lap3-2 pollen exine isthinner, weaker, and is missing some connections betweentheir roof-like tectum structures. We describe the mappingand identification of LAP3 as a novel gene that contains arepetitive motif found in b-propeller enzymes. Insertionmutations in LAP3 lead to male sterility. To investigatepossible roles for LAP3 in pollen development, we assayedthe metabolite profile of anther tissues containing developingpollen grains and found that the lap3-2 defect leadsto a broad range of metabolic changes. The largest changeswere seen in levels of a straight-chain hydrocarbon nonacosaneand in naringenin chalcone, an obligate compoundin the flavonoid biosynthesis pathway

INP1 involvement in pollen aperture formation is evolutionarily conserved and may require species-specific partners

Pollen wall exine is usually deposited non-uniformly on the pollen surface, with areas of low exine deposition corresponding to pollen apertures. Little is known about how apertures form, with the novel Arabidopsis INP1 (INAPERTURATE POLLEN1) protein currently being the only identified aperture factor. In developing pollen, INP1 localizes to three plasma membrane domains and underlies formation of three apertures. Although INP1 homologs are found across angiosperms, they lack strong sequence conservation. Thus, it has been unclear whether they also act as aperture factors and whether their sequence divergence contributes to interspecies differences in aperture patterns. To explore the functional conservation of INP1 homologs, we used mutant analysis in maize and tested whether homologs from several other species could function in Arabidopsis. Our data suggest that the INP1 involvement in aperture formation is evolutionarily conserved, despite the significant divergence of INP1 sequences and aperture patterns, but that additional species-specific factors are likely to be required to guide INP1 and to provide information for aperture patterning. To determine the regions in INP1 necessary for its localization and function, we used fragment fusions, domain swaps, and interspecific protein chimeras. We demonstrate that the central portion of the protein is particularly important for mediating the species-specific functionality.Funding was provided to AAD by the US National Science Foundation (MCB-1517511) and to VNSS by the Spanish Ministry of Economy and Competitiveness (CGL2015-70290-P). PL was supported by the China Scholarship Council. SB-MS was supported by the University of Granada, Spain (grant Cei BioTic). We thank the Arabidopsis Biological Resource Center (OSU) and the Maize Genetics Cooperation Stock Center (USDA/ ARS) for seed stocks, Priscila Rodriguez Garcia (OSU) for help with characterizing Arabidopsis–tomato INP1 chimeras, and Jay Hollick (OSU) for advice on all things maize

LAP3, a novel plant protein required for pollen development, is essential for proper exine formation

We isolated lap3-1 and lap3-2 mutants in ascreen for pollen that displays abnormal stigma binding.Unlike wild-type pollen, lap3-1 and lap3-2 pollen exine isthinner, weaker, and is missing some connections betweentheir roof-like tectum structures. We describe the mappingand identification of LAP3 as a novel gene that contains arepetitive motif found in b-propeller enzymes. Insertionmutations in LAP3 lead to male sterility. To investigatepossible roles for LAP3 in pollen development, we assayedthe metabolite profile of anther tissues containing developingpollen grains and found that the lap3-2 defect leadsto a broad range of metabolic changes. The largest changeswere seen in levels of a straight-chain hydrocarbon nonacosaneand in naringenin chalcone, an obligate compoundin the flavonoid biosynthesis pathway

A Ploidy-Sensitive Mechanism Regulates Aperture Formation on the Arabidopsis Pollen Surface and Guides Localization of the Aperture Factor INP1

<div><p>Pollen presents a powerful model for studying mechanisms of precise formation and deposition of extracellular structures. Deposition of the pollen wall exine leads to the generation of species-specific patterns on pollen surface. In most species, exine does not develop uniformly across the pollen surface, resulting in the formation of apertures–openings in the exine that are species-specific in number, morphology and location. A long time ago, it was proposed that number and positions of apertures might be determined by the geometry of tetrads of microspores–the precursors of pollen grains arising via meiotic cytokinesis, and by the number of last-contact points between sister microspores. We have tested this model by characterizing Arabidopsis mutants with ectopic apertures and/or abnormal geometry of meiotic products. Here we demonstrate that contact points <i>per se</i> do not act as aperture number determinants and that a correct geometric conformation of a tetrad is neither necessary nor sufficient to generate a correct number of apertures. A mechanism sensitive to pollen ploidy, however, is very important for aperture number and positions and for guiding the aperture factor INP1 to future aperture sites. In the mutants with ectopic apertures, the number and positions of INP1 localization sites change depending on ploidy or ploidy-related cell size and not on INP1 levels, suggesting that sites for aperture formation are specified before INP1 is brought to them.</p></div

Effect of Aperture Number on Pollen Germination, Survival and Reproductive Success in \u3ci\u3eArabidopsis thaliana\u3c/i\u3e

Background and Aims Pollen grains of flowering plants display a fascinating diversity of forms, including diverse patterns of apertures, the specialized areas on the pollen surface that commonly serve as the sites of pollen tube initiation and, therefore, might play a key role in reproduction. Although many aperture patterns exist in angiosperms, pollen with three apertures (triaperturate) constitutes the predominant pollen type found in eudicot species. The aim of this study was to explore whether having three apertures provides selective advantages over other aperture patterns in terms of pollen survival, germination and reproductive success, which could potentially explain the prevalence of triaperturate pollen among eudicots. Methods The in vivo pollen germination, pollen tube growth, longevity and competitive ability to sire seeds were compared among pollen grains of Arabidopsis thaliana with different aperture numbers. For this, an arabidopsis pollen aperture series was used, which included the triaperturate wild type, as well as mutants without an aperture (inaperturate) and with more than three apertures. Key Results Aperture number appears to influence pollen grain performance. In most germination and longevity experiments, the triaperturate and inaperturate pollen grains performed better than pollen with higher aperture numbers. In mixed pollinations, in which triaperturate and inaperturate pollen were forced to compete with each other, the triaperturate pollen outperformed the inaperturate pollen. Conclusions Triaperturate pollen grains might provide the best trade-off among various pollen performance traits, thus explaining the prevalence of this morphological trait in the eudicot clade

Diploid pollen in <i>osd1</i> and <i>tam-2</i> mutants has predominantly four or six apertures, some of which develop without any contact with intersporal callose wall.

<p>(A, B’) Front and back views of <i>osd1</i> diploid pollen with four (A, A’) or six (B, B’) apertures. (C-D’) Front and back views of <i>tam-2</i> diploid pollen with four (C, C’) or six (D, D’) apertures. (E) 3D reconstruction of a diploid <i>osd1</i> dyad at the stage when developing apertures become visible on the surfaces of microspores. Two apertures that develop at the distal side of one of the microspores, away from the intersporal callose wall (CW), are indicated by arrowheads. Microspore surfaces were stained with DAPI and callose wall was stained with calcofluor white (both blue). (F-I) Dyads of mature pollen from the <i>osd1; qrt1</i> plants showing examples of dyads with 4/4 (F), 6/6 (G), 8/6 (H), and 4/6 (I) aperture configurations. Scale bars = 10 μm.</p

Pollen ploidy higher than 2n is accompanied by changes in aperture morphology.

<p>(A-F’) Ring-shaped apertures are common in tetraploid pollen from <i>osd1</i> (A-B’), <i>tam-2</i> (C- D’), and <i>tes</i> (E-F’). Front and back views are shown for each pollen grain (e.g. A and A’). (G-H’) Tetraploid pollen from <i>tam-2; osd1</i> mutants often has abnormal exine patterns that make recognizing apertures difficult. Still, ring-shaped apertures are sometimes visible in <i>tam-2; osd1</i> pollen (H, H’, arrowheads). Scale bars = 10 μm.</p

Pollen grains of the 4n plants generated from the 2n lines with reduced levels of <i>INP1</i> expression and shortened apertures develop four shortened apertures.

<p>(A) Schematic description of the experiment to distinguish between possible mechanisms involved in specifying aperture number. Tetraploid plants generated from the diploid short-aperture line by colchicine treatment are expected to produce 2n pollen with 3 longer apertures if INP1 level is the factor that determines the number of apertures. 2n pollen with 4 short apertures is expected if other factors (e.g. levels of another gene or changes in cell geometry) dictate aperture number. (B-C’) An <i>INP1pr</i>::<i>INP1-myc; inp1 S</i> line produces three short apertures in haploid pollen (B-B’) and four short apertures in diploid pollen (C-C’). An <i>INP1pr</i>::<i>INP1-myc; inp1 M</i> line produces three medium-size apertures in haploid pollen (D-D’) and four medium-size apertures in diploid pollen (E-E’). Apertures are indicated by arrowheads. Scale bars = 10 μm.</p