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.

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.

Pollen wall degradation in the Brassicaceae permits cell emergence after pollination.

PREMISE OF THE STUDY: Despite attempts to degrade the sporopollenin in pollen walls, this material has withstood a hundred years of experimental treatments and thousands of years of environmental attack in insects and soil. We present evidence that sporopollenin, nonetheless, locally degrades only minutes after pollination in Arabidopsis thaliana flowers, and describe here a two-part pollen germination mechanism in A. thaliana involving both chemical weakening of the exine wall and swelling of the underlying intine. METHODS: We explored naturally occurring components from pollen and stigma surfaces and found a tripartite mix of hydrogen peroxide, peroxidase and catalase enzymes (all at high levels at the pollination interface) to be experimentally sufficient to degrade the sporopollenin of some Brassicaceae family members. KEY RESULTS: At pollination, factors carried on the pollen surface may mix with factors on the stigma surface in a reaction that locally oxidizes the exine pollen wall. Hydrogen peroxide, catalases, and peroxidases are biologically present at the right time and place and, when mixed experimentally, are sufficient to degrade the walls of susceptible pollen. CONCLUSIONS: Our work on native biochemistry for breaching sporopollenin suggests new research directions in pollen aperture evolution and could aid efforts to analyze sporopollenin's composition, needed for application of this corrosion-resistant, but long-intractable material.

Control of Anther Cell Differentiation by the Small Protein Ligand TPD1 and Its Receptor EMS1 in Arabidopsis.

A fundamental feature of sexual reproduction in plants and animals is the specification of reproductive cells that conduct meiosis to form gametes, and the associated somatic cells that provide nutrition and developmental cues to ensure successful gamete production. The anther, which is the male reproductive organ in seed plants, produces reproductive microsporocytes (pollen mother cells) and surrounding somatic cells. The microsporocytes yield pollen via meiosis, and the somatic cells, particularly the tapetum, are required for the normal development of pollen. It is not known how the reproductive cells affect the differentiation of these somatic cells, and vice versa. Here, we use molecular genetics, cell biological, and biochemical approaches to demonstrate that TPD1 (TAPETUM DETERMINANT1) is a small secreted cysteine-rich protein ligand that interacts with the LRR (Leucine-Rich Repeat) domain of the EMS1 (EXCESS MICROSPOROCYTES1) receptor kinase at two sites. Analyses of the expressions and localizations of TPD1 and EMS1, ectopic expression of TPD1, experimental missorting of TPD1, and ablation of microsporocytes yielded results suggesting that the precursors of microsporocyte/microsporocyte-derived TPD1 and pre-tapetal-cell-localized EMS1 initially promote the periclinal division of secondary parietal cells and then determine one of the two daughter cells as a functional tapetal cell. Our results also indicate that tapetal cells suppress microsporocyte proliferation. Collectively, our findings show that tapetal cell differentiation requires reproductive-cell-secreted TPD1, illuminating a novel mechanism whereby signals from reproductive cells determine somatic cell fate in plant sexual reproduction.

Physicochemical stability of phospholipid-dispersed suspensions of crystalline itraconazole.

The physicochemical stability of an aqueous, phospholipid-based dispersion of itraconazole microcrystals was studied as a model water-insoluble drug suspension. The particle size, phospholipid concentrations, free fatty acid (FFA) content, pH, and zeta potential of two test suspensions were followed over 63 days at 5 and 40 degrees C storage conditions. Hydrolysis of a control suspension containing Lipoid E80 led to rapid FFA formation, pH drop, and subsequent particle aggregation. In the second suspension, sodium oleate used in conjunction with Lipoid E80 significantly enhanced the suspension physicochemical stability. Oleate anions effectively (1) increased the anionic charge of the phospholipid surface layer, (2) buffered the suspension near pH 7, and (3) reduced the specific production of oleic acid as a phosphatidylcholine (PC) degradant. The observed hydrolysis rate constants k(obs) approximately 2 x 10(-7) (Lipoid only) and k(obs) approximately 5 x 10(-8) (Lipoid and oleate) were consistent with the pH dependent behavior reported for saturated soybean PC solutions. Mechanistically, FFA formed initially in the control suspension partitioned to the aqueous phase with limited influence on the phospholipid microenvironment at the itraconazole particle surface. Phospholipid stabilization of water-insoluble drugs was demonstrated with clear benefits from fatty acid anions as co-additives to influence the surface microenvironment, reduce hydrolysis kinetics, and enhance suspension physicochemical stability.