Exine Wall Research Articles

PALYNOLOGY OF SOME SPECIES IN THE FAMILY CUCURBITACEAE FROM NIGERIA

Pollen morphology of ten (10) species representing nine (9) genera, two (2) varieties in the family Cucurbitaceae from Nigeria has been examined under light microscope. The pollen grains are largely monads. Exine wall ornamentation is most often reticulate in all the species of the family studied with the exception of pollens in Cucurbita maxima Duch. which are echinate, this is a diagnostic character for this species. Cell wall thickness is the least variable among all the characters studied. Almost all types of pollen shape classes have been observed within this family, namely spheroidal, subprolate, oblate-spheroidal, prolate-spheroidal, suboblate and prolate, with more than one shape type occurring in a species. It is worthy of note that pollen shape and aperture type can be used to delimit Lagenaria siceraria (spatulate fruit shape) from Lagenaria siceraria (oval fruit shape). However, the shapes are the same in Citrullus lanatus (black seeds water melon) and Citrullus lanatus (brown seeds water melon). Three (3) types of apertures, porate, colpate (very occasionally) and colporate are documented for the family in this study, while seven (7) pollen types have been distinguished based on the apertures: monoporate, monocolpate, bicolporate, triporate, tricolporate, tetraporate and tetracolporate. Tricolporate pollens were observed to occur more frequently than other apertural types. Pollen grains belong to the size categories Media and Magna and only very occasionally in the Minuta category. The implication of these size categories in the reproductive success of the plants in this family is highlighted.

Degradation of de-esterified pctin/homogalacturonan by the polygalacturonase GhNSP is necessary for pollen exine formation and male fertility in cotton.

SummaryThe pollen wall exine provides a protective layer for the male gametophyte and is largely composed of sporopollenin, which comprises fatty acid derivatives and phenolics. However, the biochemical nature of the external exine is poorly understood. Here, we show that the male sterile line 1355A of cotton mutated in NO SPINE POLLEN (GhNSP) leads to defective exine formation. The GhNSP locus was identified through map‐based cloning and confirmed by genetic analysis (co‐segregation test and allele prediction using the CRISPR/Cas9 system). In situ hybridization showed that GhNSP is highly expressed in tapetum. GhNSP encodes a polygalacturonase protein homologous to AtQRT3, which suggests a function for polygalacturonase in pollen exine formation. These results indicate that GhNSP is functionally different from AtQRT3, the latter has the function of microspore separation. Biochemical analysis showed that the percentage of de‐esterified pectin was significantly increased in the 1355A anthers at developmental stage 8. Furthermore, immunofluorescence studies using antibodies to the de‐esterified and esterified homogalacturonan (JIM5 and JIM7) showed that the Ghnsp mutant exhibits abundant of de‐esterified homogalacturonan in the tapetum and exine, coupled with defective exine formation. The characterization of GhNSP provides new understanding of the role of polygalacturonase and de‐esterified homogalacturonan in pollen exine formation.

DCET1 Controls Male Sterility Through Callose Regulation, Exine Formation, and Tapetal Programmed Cell Death in Rice.

In angiosperms, anther development comprises of various complex and interrelated biological processes, critically needed for pollen viability. The transitory callose layer serves to separate the meiocytes. It helps in primexine formation, while the timely degradation of tapetal cells is essential for the timely callose wall dissolution and pollen wall formation by providing nutrients for pollen growth. In rice, many genes have been reported and functionally characterized that are involved in callose regulation and pollen wall patterning, including timely programmed cell death (PCD) of the tapetum, but the mechanism of pollen development largely remains ambiguous. We identified and functionally characterized a rice mutant dcet1, having a complete male-sterile phenotype caused by defects in anther callose wall, exine patterning, and tapetal PCD. DCET1 belongs to the RNA recognition motif (RRM)-containing family also called as the ribonucleoprotein (RNP) domain or RNA-binding domain (RBD) protein, having single-nucleotide polymorphism (SNP) substitution from G (threonine-192) to A (isoleucine-192) located at the fifth exon of LOC_Os08g02330, was responsible for the male sterile phenotype in mutant dcet1. Our cytological analysis suggested that DCET1 regulates callose biosynthesis and degradation, pollen exine formation by affecting exine wall patterning, including abnormal nexine, collapsed bacula, and irregular tectum, and timely PCD by delaying the tapetal cell degeneration. As a result, the microspore of dcet1 was swollen and abnormally bursted and even collapsed within the anther locule characterizing complete male sterility. GUS and qRT-PCR analysis indicated that DCET1 is specifically expressed in the anther till the developmental stage 9, consistent with the observed phenotype. The characterization of DCET1 in callose regulation, pollen wall patterning, and tapetal cell PCD strengthens our knowledge for knowing the regulatory pathways involved in rice male reproductive development and has future prospects in hybrid rice breeding.

Glucuronidation of type II arabinogalactan polysaccharides function in sexual reproduction of Arabidopsis.

Arabinogalactan proteins (AGPs) are complex, hyperglycosylated plant cell wall proteins with little known about the biological roles of their glycan moieties in sexual reproduction. Here, we report that GLCAT14A, GLCAT14B, and GLCAT14C, three enzymes responsible for the addition of glucuronic acid residues to AGPs, function in pollen development, polytubey block, and normal embryo development in Arabidopsis. Using biochemical and immunolabeling techniques, we demonstrated that the loss of function of the GLCAT14A, GLCAT14B, and GLCAT14C genes resulted in disorganization of the reticulate structure of the exine wall, abnormal development of the intine layer, and collapse of pollen grains in glcat14a/b and glcat14a/b/c mutants. Synchronous development between locules within the same anther was also lost in some glcat14a/b/c stamens. In addition, we observed excessive attraction of pollen tubes targeting glcat14a/b/c ovules, indicating that the polytubey block mechanism was compromised. Monosaccharide composition analysis revealed significant reductions in all sugars in glcat14a/b and glcat14a/b/c mutants except for arabinose and galactose, while immunolabeling showed decreased amounts of AGP sugar epitopes recognized by glcat14a/b and glcat14a/b/c mutants compared with the wild type. This work demonstrates the important roles that AG glucuronidation plays in Arabidopsis sexual reproduction and reproductive development.

Members of the ELMOD protein family specify formation of distinct aperture domains on the Arabidopsis pollen surface.

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.

Dynamic changes in primexine during the tetrad stage of pollen development.

Formation of pollen wall exine is preceded by the development of several transient layers of extracellular materials deposited on the surface of developing pollen grains. One such layer is primexine (PE), a thin, ephemeral structure that is present only for a short period of time and is difficult to visualize and study. Recent genetic studies suggested that PE is a key factor in the formation of exine, making it critical to understand its composition and the dynamics of its formation. In this study, we used high-pressure frozen/freeze-substituted samples of developing Arabidopsis (Arabidopsis thaliana) pollen for a detailed transmission electron microscopy analysis of the PE ultrastructure throughout the tetrad stage of pollen development. We also analyzed anthers from wild-type Arabidopsis and three mutants defective in PE formation by immunofluorescence, carefully tracing several carbohydrate epitopes in PE and nearby anther tissues during the tetrad and the early free-microspore stages. Our analyses revealed likely sites where these carbohydrates are produced and showed that the distribution of these carbohydrates in PE changes significantly during the tetrad stage. We also identified tools for staging tetrads and demonstrate that components of PE undergo changes resembling phase separation. Our results indicate that PE behaves like a much more dynamic structure than has been previously appreciated and clearly show that Arabidopsis PE creates a scaffolding pattern for formation of reticulate exine.

Differences in the structure of pollen in the ‘Senga Sengana’ and ‘Selva’ cultivars of Fragaria × ananasa

The strawberry belongs to one of the youngest species of arable crops. The study aimed to analyze pollen's structure in the ‘Senga Sengana’ and ‘Selva’ cultivars of Fragaria × ananassa. The strawberry is a dicotyledonous plant from the Rosaceae family. The article presents research carried out on Fragaria × ananassa cv. ‘Senga Sengana’ and ‘Selva’ collected during flowering. Pollen mor-phology was examined using a scanning electron microscope and a traditional optical microscope. The results showed that fresh pollen outnumbered dead pollen, confirming that the ‘Senga Sengana’ culti-var produces larger amounts of fertile pollen. Mature ‘Senga Sengana’ pollen grains are characterized by a specific sculpture of the exine walls. The analysis of Selva pollen grains revealed substantial changes in the structure of the sporoderm during its maturation. It becomes thicker and, as the size of the pollen grain increases, the exine layer becomes more elaborate.

Naturally occurring triploid birch hybrids from woodlands in Iceland are partially fertile

Woodlands in Iceland are characterized by shrub-like birch, a result of introgressive hybridization between diploid dwarf birch, Betula nana, and tetraploid downy birch, B. pubescens. Introgression occurs via triploid hybrids. We evaluated the fertility of triploid birch by examining pollen viability and seed germination. Pollen samples were collected from woodlands throughout Iceland from 99 plants, including 22 triploid hybrids. The germination of pollen from the triploid hybrids was about one third that of the diploid or tetraploid species (mean values: 9% vs. 26% and 29%). Most triploid plants had a pollen germination of less than 2%. However, some showed exceptionally high pollen fertility (six plants: 11–79%). Microscopic examination revealed normal pollen tube growth and elongation in hybrids with low germination, while other hybrids showed abnormal tube growth. Fluorescence microscopy indicated pollen viability in all ploidy groups, with green autofluorescence from the cytoplasmic contents and red fluorescence from the exine wall. The Aniline test of pollen viability showed that grains with three pores (normal, triporate grains) stained positively, but non-triporate grains were unstained and empty. Germination tests were performed on seeds collected from 246 plants, including 21 triploid hybrids. The germination percentage of seeds from triploid hybrid plants was about 20 times lower than that of the diploid or the tetraploid species (population means: 0–4% vs. 3–41% and 6–54%). The present study shows that triploid birch hybrids are not sterile. Both the paternal and maternal fertility of the hybrids should be sufficient to facilitate gene flow via backcrossing with the parental species.

Mechanical design of apertures and the infolding of pollen grain

When pollen grains become exposed to the environment, they rapidly desiccate. To protect themselves until rehydration, the grains undergo characteristic infolding with the help of special structures in the grain wall-apertures-where the otherwise thick exine shell is absent or reduced in thickness. Recent theoretical studies have highlighted the importance of apertures for the elastic response and the folding of the grain. Experimental observations show that different pollen grains sharing the same number and type of apertures can nonetheless fold in quite diverse fashions. Using the thin-shell theory of elasticity, we show how both the absolute elastic properties of the pollen wall and the relative elastic differences between the exine wall and the apertures play an important role in determining pollen folding upon desiccation. Focusing primarily on colpate pollen, we delineate the regions of pollen elastic parameters where desiccation leads to a regular, complete closing of all apertures and thus to an infolding which protects the grain against water loss. Phase diagrams of pollen folding pathways indicate that an increase in the number of apertures leads to a reduction of the region of elastic parameters where the apertures close in a regular fashion. The infolding also depends on the details of the aperture shape and size, and our study explains how the features of the mechanical design of apertures influence the pollen folding patterns. Understanding the mechanical principles behind pollen folding pathways should also prove useful for the design of the elastic response of artificial inhomogeneous shells.

Pollen Grains Morphology of Angiosperms

Pollen morphology of fourteen species of angiosperms from Kathmandu valley was investigated using aceto-carmine and palynological characters such as pollen size, shape, aperture numbers and exine sculpture were evaluated. The results indicate that various types of pollen grains are in angiosperms. In this investigation Pollen grains of Linum usitatissimum L. from the family Linaceae, Lathyrus odoratus L. from Fabaceae, Magnolia grandiflora L. from Magnoliaceae, Malva alcea L. and Malva sida L. from Malvaceae, Murraya koenigii Spreng from Rutaceae, Nerium oleander L. from Apocynaceae studied. Likewise, Ocimum tenuiflorum L., Salvia coccinea Buchz ex Etl. Salvia splendens Sellow ex J.A. Schultes from Lamiaceae, Oenothera rosea L. from Onagraceae, Prunus persica (L.) Batsch. from Rosaceae, Solanum nigrum L. from Solanaceae and Zinnia elegans L. from Asteraceae were studied in this investigation. Shape of the pollen grains found to be spheroidal, sub-spheroidal, elongate, oval, circular, ellipsoidal and triangular type. Ornamentation of exine wall found to be echinate, smooth, coarse, wrinkle and tected type. Aperture of the pollen grains found to be triporate to periporate. Sizes of the pollen grains encountered in this study were large, medium and small. The high diversity of exine ornamentation type in pollen grains of angiosperms has been associated to diversity in pollination systems. Echinate pollen grains train the bees to restrict to nectar collection and help the flowers to save more pollen grains for pollination. Smooth walled pollen grains are linked with wind or water pollination.
 Int. J. Appl. Sci. Biotechnol. Vol 8(2): 205-210

The temporal regulation of TEK contributes to pollen wall exine patterning.

Pollen wall consists of several complex layers which form elaborate species-specific patterns. In Arabidopsis, the transcription factor ABORTED MICROSPORE (AMS) is a master regulator of exine formation, and another transcription factor, TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK), specifies formation of the nexine layer. However, knowledge regarding the temporal regulatory roles of TEK in pollen wall development is limited. Here, TEK-GFP driven by the AMS promoter was prematurely expressed in the tapetal nuclei, leading to complete male sterility in the pAMS:TEK-GFP (pat) transgenic lines with the wild-type background. Cytological observations in the pat anthers showed impaired callose synthesis and aberrant exine patterning. CALLOSE SYNTHASE5 (CalS5) is required for callose synthesis, and expression of CalS5 in pat plants was significantly reduced. We demonstrated that TEK negatively regulates CalS5 expression after the tetrad stage in wild-type anthers and further discovered that premature TEK-GFP in pat directly represses CalS5 expression through histone modification. Our findings show that TEK flexibly mediates its different functions via different temporal regulation, revealing that the temporal regulation of TEK is essential for exine patterning. Moreover, the result that the repression of CalS5 by TEK after the tetrad stage coincides with the timing of callose wall dissolution suggests that tapetum utilizes temporal regulation of genes to stop callose wall synthesis, which, together with the activation of callase activity, achieves microspore release and pollen wall patterning.

Morphological Study of Pollen Grains of Angiosperms

Pollen morphology of twelve species of angiosperms from Central Nepal was cytologically carried out in present investigation. In this investigation pollen morphology of Bignonia venusta Ker Gawl. and Jacranda mimisifolia D.Don from the family Bignoniaceae; Brassica compestris L. from Brassicaceae; Cuphea hyssopifolia Kunth. from Lythraceae; Coriandrum sativum L. from Apiaceae; Crepis japonica L. and Dichrocephala integrifolia (L f.) kunzte. from Asteraceae; Delphinium ajacis L. from Ranunculaceae; Dianthus barbetus L. from Caryophyllaceae; Euphorbia Milii Des Moul. and Euphorbia pulcherima L. from Euphorbiaceae and Magnolia fuscata from Magnoliaceae were studied. Shape of the pollen grains found to be mostly spheroidal, few circular, one elliptical, one triangular and one elongated in this study. Sculpture of exine wall found to be smooth, echidnae, tected, reticulated and perforated type. Aperture of the pollen grains found to be triporate to pentaporate. The large, medium and small sized pollen grains were observed in present research. The high diversty of sculpture type in pollen grains of angiosperms has been associated to diversity in pollination systems. Smooth pollen grains are linked with wind or water pollination while sculptured pollen grains associated with biotic pollination. The morphology of pollen grain is one of the significant tools in solving some taxonomic problems such as identification, tracing phylogenetic relationship on the family, generic or specific level and in plant systematic and evolution.
 Int. J. Appl. Sci. Biotechnol. Vol 7(3): 354-358

Changes in morphogen kinetics and pollen grain size are potential mechanisms of aberrant pollen aperture patterning in previously observed and novel mutants of Arabidopsis thaliana.

Pollen provides an excellent system to study pattern formation at the single-cell level. Pollen surface is covered by the pollen wall exine, whose deposition is excluded from certain surface areas, the apertures, which vary between the species in their numbers, positions, and morphology. What determines aperture patterns is not understood. Arabidopsis thaliana normally develops three apertures, equally spaced along the pollen equator. However, Arabidopsis mutants whose pollen has higher ploidy and larger volume develop four or more apertures. To explore possible mechanisms responsible for aperture patterning, we developed a mathematical model based on the Gierer-Meinhardt system of equations. This model was able to recapitulate aperture patterns observed in the wild-type and higher-ploidy pollen. We then used this model to further explore geometric and kinetic factors that may influence aperture patterns and found that pollen size, as well as certain kinetic parameters, like diffusion and decay of morphogens, could play a role in formation of aperture patterns. In conjunction with mathematical modeling, we also performed a forward genetic screen in Arabidopsis and discovered two mutants with aperture patterns that had not been previously observed in this species but were predicted by our model. The macaron mutant develops a single ring-like aperture, matching the unusual ring-like pattern produced by the model. The doughnut mutant forms two pore-like apertures at the poles of the pollen grain. Further tests on these novel mutants, motivated by the modeling results, suggested the existence of an area of inhibition around apertures that prevents formation of additional apertures in their vicinity. This work demonstrates the ability of the theoretical model to help focus experimental efforts and to provide fundamental insights into an important biological process.

A proteomic study of cysteine protease induced cell death in anthers of male sterile tobacco transgenic plants.

Manifestation of male sterility in plants is an important requirement for hybrid seed production. Tapetum cell layer of anther is a primary target for genetic manipulation for male sterility. In our previous report, the targeted expression of Arachis cysteine protease in tapetum led to premature degeneration of tapetal layer that resulted in complete male sterility in transgenic tobacco plants. To correlate cysteine protease mediated cell death of tapetum, transmission electron microscopy (TEM) and proteomic pattern of anthers of cysteine protease induced male sterile plant were compared with the untransformed control plant. TEM study revealed the abnormal growth of tapetal cells exhibiting excessive vacuolization that synchronized with irregular exine wall formation of the microspores. In anther proteome, a total 250 protein spots were detected that were reproducible and exhibited similar distribution pattern. Further, anther proteome of male sterile plant showed the significant upregulation (≥ 1.5) of 56 protein spots. Using Mass spectroscopy (MALDI TOF/TOF), we have identified 14 protein spots that were involved in several processes such as energy metabolism, protein synthesis, plastid protein, lipid metabolism, and cell wall assembly. Upregulation of patatin-like protein-2 homolog, carboxylesterase 17 and dicer like protein-4 in male sterile anthers that have been demonstrated to induce cell death, suggesting that cysteine protease mediated premature tapetal cell death might involve the lipid peroxidation pathway in coordination with gene silencing mechanism.

Arabidopsis Protein Kinase D6PKL3 Is Involved in the Formation of Distinct Plasma Membrane Aperture Domains on the Pollen Surface.

Certain regions on the surfaces of developing pollen grains exhibit very limited deposition of pollen wall exine. These regions give rise to pollen apertures, which are highly diverse in their patterns and specific for individual species. Arabidopsis thaliana pollen develops three equidistant longitudinal apertures. The precision of aperture formation suggests that, to create them, pollen employs robust mechanisms that generate distinct cellular domains. To identify players involved in this mechanism, we screened natural Arabidopsis accessions and discovered one accession, Martuba, whose apertures form abnormally due to the disruption of the protein kinase D6PKL3. During pollen development, D6PKL3 accumulates at the three plasma membrane domains underlying future aperture sites. Both D6PKL3 localization and aperture formation require kinase activity. Proper D6PKL3 localization is also dependent on a polybasic motif for phosphoinositide interactions, and we identified two phosphoinositides that are specifically enriched at the future aperture sites. The other known aperture factor, INAPERTURATE POLLEN1, fails to aggregate at the aperture sites in d6pkl3 mutants, changes its localization when D6PKL3 is mislocalized, and, in turn, affects D6PKL3 localization. The discovery of aperture factors provides important insights into the mechanisms cells utilize to generate distinct membrane domains, develop cell polarity, and pattern their surfaces.

Pollen morphology and trichome types of Betula spp. in the Hyrcanian forests of northern Iran

Pollen and trichome morphology of the genus Betula were evaluated from three main habitats in Iran using light and electron microscopy. Pollen types were identical and tri-porate; however, in terms of quantitative data, some differences were observed in exine (outside) wall thickness, length of polar axis and length of equatorial axis, possibly due to the environment. Pollen alone is insufficient to separate species of Iranian birch. Investigation of the trichome trait (small hairs or other outgrowths) revealed that birch species in Iran belong to: (1) Betula litwinowii Doluch. according to the low density of trichomes on the adaxial surface of the leaves and the lack of trichomes on the abaxial surface and many trichomes on the petioles; (2) Betula pendula Roth. due to the absence of trichomes on the adaxial surface and the low density of trichomes on the abaxial surface; and, (3) in the adaxial and abaxial surfaces of leaves and petioles, the trichomes had different types of pilose (long, soft hairs), and this strengthens the possibility of a different species or more likely a hybrid of B. pendula.

Pollen Dysfunction Causes ‘Floral Bud Distortion’ in Indian Soybean (Glycine max)

Floral bud distortion (FBD) is a peculiar disorder limiting yield of soybean in central India. Investigations were conducted to understand tissue-specific cytological, biochemical and molecular alterations associated with FBD. The conspicuous morphological symptom seen was extended vegetative phase, which remained green even after R7 (beginning maturity) stage of growth and failure to produce pods leading to complete yield loss in affected plants. Reduced numbers of pollen grains as well as pollens with high percent of sterility were recorded in symptomatic plants. Although the stigma was found to be receptive in the symptomatic plants, pollens showed irregular shape and had a thicker exine wall. Carbohydrate (2.21 times), protein (2.38 times) and chlorophyll (2.17 times) contents were found to be significantly higher in symptomatic plants. Tissue-specific differentially expressed TDFs (transcript-derived fragments) were generated through cDNA-RAPD using 40 primers. Seven primers were found polymorphic and size ranged from 160 to 1100 bp. Re-amplified TDFs were sequenced, and they showed partial homology with characterized (ARF, XM_003529058) and uncharacterized (LOC 100815325) transcription factors. This indicates investigating differentially expressed genes are needed to understand the insights of molecular alterations associated with floral bud distortion.

Preserving the inflated structure of lyophilized sporopollenin exine capsules with polyethylene glycol osmolyte

Extracted from natural pollen grains, sporopollenin exine capsules (SECs) are robust, chemically inert biopolymer shells that posess highly uniform size and shape characteristics and that can be utilized as hollow microcapsules for drug delivery applications. However, it is challenging to extract fully functional SECs from many pollen species because pollen grains often collapse, causing the loss of architectural features, loading volume, and bulk uniformity. Herein, we demonstrate that polyethylene glycol (PEG) osmolyte solutions can help preserve the native architectural features of extracted SECs, yielding inflated microcapsules of high uniformity that persist even after subsequent lyophilization. Optimal conditions were first identified to extract SECs from cattail (Typhae angustfolia) pollen via phosphoric acid processing after which successful protein removal was confirmed by elemental (CHN), mass spectrometry (MALDI-TOF), and confocal laser canning microscopy (CLSM) analyses. The shape of SECs was then assessed by scanning electron microscopy (SEM) and dynamic image particle analysis (DIPA). While acid-processed SECs experienced high degrees of structural collapse, incubation in 2.5% or higher PEG solutions significantly improved preservation of spherical SEC shape by inducing inflation within the microcapsules. A theoretical model of PEG-induced osmotic pressure effects was used to interpret the experimental data, and the results show excellent agreement with the known mechanical properties of pollen exine walls. Taken together, these findings demonstrate that PEG osmolyte is a useful additive for preserving particle shape in lyophilized SEC formulations, opening the door to broadly applicable strategies for stabilizing the structure of hollow microcapsules.

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.

Pollen Aperture Factor INP1 Acts Late in Aperture Formation by Excluding Specific Membrane Domains from Exine Deposition.

Accurate placement of extracellular materials is a critical part of cellular development. To study how cells achieve this accuracy, we use formation of pollen apertures as a model. In Arabidopsis (Arabidopsis thaliana), three regions on the pollen surface lack deposition of pollen wall exine and develop into apertures. In developing pollen, Arabidopsis INAPERTURATE POLLEN1 (INP1) protein acts as a marker for the preaperture domains, assembling there into three punctate lines. To understand the mechanism of aperture formation, we studied the dynamics of INP1 expression and localization and its relationship with the membrane domains at which it assembles. We found that INP1 assembly occurs after meiotic cytokinesis at the interface between the plasma membrane and the overlying callose wall, and requires the normal callose wall formation. Sites of INP1 localization coincide with positions of protruding membrane ridges in proximity to the callose wall. Our data suggest that INP1 is a late-acting factor involved in keeping specific membrane domains next to the callose wall to prevent formation of exine at these sites.