Primexine Deposition Research Articles

ADAPTOR PROTEIN-1 complex-mediated post-Golgi trafficking is critical for pollen wall development in Arabidopsis.

Summary Primexine deposition is essential for the formation of pollen wall patterns and is precisely regulated by the tapetum and microspores. While tapetum‐ and/or microspore‐localized proteins are required for primexine biosynthesis, how their trafficking is established and controlled is poorly understood.In Arabidopsis thaliana, AP1σ1 and AP1σ2, two genes encoding the σ subunit of the trans‐Golgi network/early endosome (TGN/EE)‐localized ADAPTOR PROTEIN‐1 complex (AP‐1), are partially redundant for plant viability, and the loss of AP1σ1 function reduces male fertility due to defective primexine formation. Here, we investigated the role of AP‐1 in pollen wall formation.The deposition of Acyl‐CoA SYNTHETASE5 (ACOS5) and type III LIPID TRANSFER PROTEINs (LTPs) secreted from the anther tapetum, which are involved in exine formation, were impaired in ap1σ1 mutants. In addition, the microspore plasma membrane (PM) protein RUPTURED POLLEN GRAIN1 (RPG1), which regulates primexine deposition, accumulated abnormally at the TGN/EE in ap1σ1 mutants. We show that AP‐1μ recognizes the YXXΦ motif of RPG1, thereby regulating its PM abundance through endocytic trafficking, and that loss of AP1σ1 decreases the levels of other AP‐1 subunits at the TGN/EE.Our observations show that AP‐1‐mediated post‐Golgi trafficking plays a vital role in pollen wall development by regulating protein transport in tapetal cells and microspores.

NERD1 is required for primexine formation and plasma membrane undulation during microsporogenesis in Arabidopsis thaliana.

The primexine formation and plasma membrane undulation are the crucial steps of pollen wall formation in many angiosperms. However, the molecular mechanism underlining these processes is largely unknown. In Arabidopsis, NEW ENHANCER OF ROOT DWARFISM1 (NERD1), a transmembrane protein, was reported to play pleiotropic roles in plant development including male fertility control; while, how NERD1 disruption impacts male reproduction is yet unclear. Here, we revealed that the male sterility of nerd1 mutants is attributed to defects in early steps of pollen wall formation. We found that nerd1-2 is void of primexine formation and microspore plasma membrane undulation, defective in callose deposition. Consequently, sporopollenin precursors are unable to deposit and assemble on the microspore surface, but instead accumulated in the anther locule and tapetal cells, and ultimately leading to microspore abortion. NERD1 is localized in the Golgi and is expressed in both vegetative and reproductive organs, with the highest expression in reproductive tissues, including the tapetum, male meiocytes, tetrads and mature pollen grains. Our results suggest that NERD1 is required for the primexine deposition and microspore plasma membrane undulation, thus essential for sporopollenin assembly and pollen exine formation.

Arabidopsis Cys2/His2 zinc-finger protein MAZ1 is essential for intine formation and exine pattern

Cys2/His2 zinc-finger protein (C2H2-ZFP) is widely involved in the reproductive development of plants, but its role in pollen development is still elusive. Here, we identified a pollen-related C2H2-ZFP gene named as MALE FERTILITY-ASSOCIATED ZINC FINGER PROTEIN 1 (MAZ1), which was first isolated from Arabidopsis thaliana. MAZ1 showed a preferential expression pattern in early anther development. Its mutation resulted in aberrant primexine deposition at the tetrad stage, followed by a defective multiple-layer pattern of exine with irregular baculum and no tectum. Furthermore, microspore development was arrested, and no intine layer was formed. These developmental defects led to fertility reduction and pollen abortion. This study reveals the essential role of MAZ1 in pollen wall development.

AUXIN RESPONSE FACTOR17 Is Essential for Pollen Wall Pattern Formation in Arabidopsis

In angiosperms, pollen wall pattern formation is determined by primexine deposition on the microspores. Here, we show that AUXIN RESPONSE FACTOR17 (ARF17) is essential for primexine formation and pollen development in Arabidopsis (Arabidopsis thaliana). The arf17 mutant exhibited a male-sterile phenotype with normal vegetative growth. ARF17 was expressed in microsporocytes and microgametophytes from meiosis to the bicellular microspore stage. Transmission electron microscopy analysis showed that primexine was absent in the arf17 mutant, which leads to pollen wall-patterning defects and pollen degradation. Callose deposition was also significantly reduced in the arf17 mutant, and the expression of CALLOSE SYNTHASE5 (CalS5), the major gene for callose biosynthesis, was approximately 10% that of the wild type. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that ARF17 can directly bind to the CalS5 promoter. As indicated by the expression of DR5-driven green fluorescent protein, which is an synthetic auxin response reporter, auxin signaling appeared to be specifically impaired in arf17 anthers. Taken together, our results suggest that ARF17 is essential for pollen wall patterning in Arabidopsis by modulating primexine formation at least partially through direct regulation of CalS5 gene expression.

Arabidopsis RPG1 is important for primexine deposition and functions redundantly with RPG2 for plant fertility at the late reproductive stage

Arabidopsis Ruptured Pollen Grain-1 (RPG1/Sweet8) is a member of the MtN3/saliva protein family that functions as a sugar transporter. The rpg1 mutant shows defective exine pattern formation. In this study, transmission electron microscopy (TEM) observations showed that much less primexine was deposited in rpg1 tetrads. Furthermore, microspore membrane undulation was abnormal, and sporopollenin accumulation was also defective. This suggests that a reduced primexine deposition in rpg1 leads to abnormal membrane undulation that affects exine pattern formation. Chemical staining revealed thinning of the callose wall of rpg1, as well as significantly reduced expression of Callose synthase-5 (CalS5) in rpg1. The fertility of the rpg1 mutant could be partly restored at late reproductive stages, potentially complemented in part by RPG2, another member of the MtN3/saliva family, which is expressed in the anther during microsporogenesis. The double mutant, rpg1rpg2, was almost sterile and was not restored during late reproduction. These results suggest that RPG1 and RPG2 are involved in primexine deposition and therefore pollen wall pattern formation.

NO PRIMEXINE AND PLASMA MEMBRANE UNDULATION Is Essential for Primexine Deposition and Plasma Membrane Undulation during Microsporogenesis in Arabidopsis

Primexine deposition and plasma membrane undulation are the initial steps of pollen wall formation. However, little is known about the genes involved in this important biological process. Here, we report a novel gene, NO PRIMEXINE AND PLASMA MEMBRANE UNDULATION (NPU), which functions in the early stage of pollen wall development in Arabidopsis (Arabidopsis thaliana). Loss of NPU function causes male sterility due to a defect in callose synthesis and sporopollenin deposition, resulting in disrupted pollen in npu mutants. Transmission electronic microscopy observation demonstrated that primexine deposition and plasma membrane undulation are completely absent in the npu mutants. NPU encodes a membrane protein with two transmembrane domains and one intracellular domain. In situ hybridization analysis revealed that NPU is strongly expressed in microspores and the tapetum during the tetrad stage. All these results together indicate that NPU plays a vital role in primexine deposition and plasma membrane undulation during early pollen wall development.

RUPTURED POLLEN GRAIN1, a Member of the MtN3/saliva Gene Family, Is Crucial for Exine Pattern Formation and Cell Integrity of Microspores in Arabidopsis

During microsporogenesis, the microsporocyte (or microspore) plasma membrane plays multiple roles in pollen wall development, including callose secretion, primexine deposition, and exine pattern determination. However, plasma membrane proteins that participate in these processes are still not well known. Here, we report that a new gene, RUPTURED POLLEN GRAIN1 (RPG1), encodes a plasma membrane protein and is required for exine pattern formation of microspores in Arabidopsis (Arabidopsis thaliana). The rpg1 mutant exhibits severely reduced male fertility with an otherwise normal phenotype, which is largely due to the postmeiotic abortion of microspores. Scanning electron microscopy examination showed that exine pattern formation in the mutant is impaired, as sporopollenin is randomly deposited on the pollen surface. Transmission electron microscopy examination further revealed that the primexine formation of mutant microspores is aberrant at the tetrad stage, which leads to defective sporopollenin deposition on microspores and the locule wall. In addition, microspore rupture and cytoplasmic leakage were evident in the rpg1 mutant, which indicates impaired cell integrity of the mutant microspores. RPG1 encodes an MtN3/saliva family protein that is integral to the plasma membrane. In situ hybridization analysis revealed that RPG1 is strongly expressed in microsporocyte (or microspores) and tapetum during male meiosis. The possible role of RPG1 in microsporogenesis is discussed.

DEX1, a Novel Plant Protein, Is Required for Exine Pattern Formation during Pollen Development in Arabidopsis

To identify factors that are required for proper pollen wall formation, we have characterized the T-DNA-tagged, dex1 mutation of Arabidopsis, which results in defective pollen wall pattern formation. This study reports the isolation and molecular characterization of DEX1 and morphological and ultrastructural analyses of dex1 plants. DEX1 encodes a novel plant protein that is predicted to be membrane associated and contains several potential calcium-binding domains. Pollen wall development in dex1 plants parallels that of wild-type plants until the early tetrad stage. In dex1 plants, primexine deposition is delayed and significantly reduced. The normal rippling of the plasma membrane and production of spacers observed in wild-type plants is also absent in the mutant. Sporopollenin is produced and randomly deposited on the plasma membrane in dex1 plants. However, it does not appear to be anchored to the microspore and forms large aggregates on the developing microspore and the locule walls. Based on the structure of DEX1 and the phenotype of dex1 plants, several potential roles for the protein are proposed.

DEX1, a Novel Plant Protein, Is Required for Exine Pattern Formation during Pollen Development in Arabidopsis

To identify factors that are required for proper pollen wall formation, we have characterized the T-DNA-tagged, dex1mutation of Arabidopsis, which results in defective pollen wall pattern formation. This study reports the isolation and molecular characterization of DEX1 and morphological and ultrastructural analyses of dex1 plants.DEX1 encodes a novel plant protein that is predicted to be membrane associated and contains several potential calcium-binding domains. Pollen wall development in dex1 plants parallels that of wild-type plants until the early tetrad stage. Index1 plants, primexine deposition is delayed and significantly reduced. The normal rippling of the plasma membrane and production of spacers observed in wild-type plants is also absent in the mutant. Sporopollenin is produced and randomly deposited on the plasma membrane in dex1 plants. However, it does not appear to be anchored to the microspore and forms large aggregates on the developing microspore and the locule walls. Based on the structure of DEX1 and the phenotype of dex1 plants, several potential roles for the protein are proposed.

Development of the echinate pollen wall inFarfugium japonicum (Compositae: Senecioneae)

Development of the echinate pollen grains inFarfugium (Compositae: Senecioneae) has been studied by a combination of transmission electron microscopy and field emission scanning electron microscopy with a freeze fractured method. The inner surface of the callose wall surrounding each microspore does not possess an echinate pattern before primexine deposition begins. The primexine formation coincides with the initiation of spines. The freeze fractured primexine shows probacula which form transverse rods. The developing exine has an inner spongy substructure. The endexine is formed by the accumulation of the electron dense lamellae with white lines after the dissolution of the callose wall. In the present study, it is confirmed that the developmental process of pollen formation revealed in the field emission scanning electron microscope is consistent with the results obtained using the transmission electron microscope.

Pollen wall morphogenesis in Tragopogon porrifolius L. (Compositae: Lactuceae) and its taxonomic significance

Pollen development in Tragopogon porrifolius has been studied in the scanning electron microscope to establish the origin of taxonomically significant features. As in other members of the tribe recently investigated the echinolophate condition is shown to result from differential deposition of the tetrad callose wall and, subsequently, of primexine. Tragopogon differs from other Lactuceae in the absence of tectum and columellae in the lacunae, this also appears to be the result of differential primexine deposition. The development of onci and their extrusion at the apertures are described. The contribution of developmental studies of pollen grains to systematics is discussed.

Male Sterility in Soybean (Glycine max). I. Phenotypic Expression of the ms2 Mutant

The effects of a nuclear male-sterile mutant (ms2) of soybean, Glycine max (L.) Merr., on anther development were analyzed by means of light- and electron-microscopy. The structure of microspore mother cells (MMCs) in male-sterile plants was identical to that of male-fertile plants. Meiosis was completed, and tetrads of microspores formed. Microspores degenerated after the deposition of primexine and probacullae. The sheath of callose surrounding microspores did not dissolve. No structural abnormalities of the microspores were detected before the onset of degeneration. The tapetal and anther wall layers were characterized by aberrant development. Tapetal abnormalities included premature vacuolation, a persistent inner tangential cell wall, failure to differentiate normal concentrations of endoplasmic reticulum and dictyosomes, disruption of plastids, and premature degeneration. Malfunction of the tapetal layer preceded, and may have induced, microspore degeneration. Gross anther morphology was not influenced until advanced stages of development.

MALE STERILITY IN SOYBEAN (GLYCINE MAX). I. PHENOTYPIC EXPRESSION OF THE ms2 MUTANT

The effects of a nuclear male‐sterile mutant (ms2) of soybean, Glycine max (L.) Merr., on anther development were analyzed by means of light‐ and electron‐microscopy. The structure of microspore mother cells (MMCs) in male‐sterile plants was identical to that of male‐fertile plants. Meiosis was completed, and tetrads of microspores formed. Microspores degenerated after the deposition of primexine and probacullae. The sheath of callose surrounding microspores did not dissolve. No structural abnormalities of the microspores were detected before the onset of degeneration. The tapetal and anther wall layers were characterized by aberrant development. Tapetal abnormalities included premature vacuolation, a persistent inner tangential cell wall, failure to differentiate normal concentrations of endoplasmic reticulum and dictyosomes, disruption of plastids, and premature degeneration. Malfunction of the tapetal layer preceded, and may have induced, microspore degeneration. Gross anther morphology was not influenced until advanced stages of development.

A comparative light and electron microscopic study of microsporogenesis in male-fertile and cytoplasmic male-sterile pepper (Capsicum annuum)

In the male-fertile line of pepper, microsporogenesis and pollen development are normal. During meiosis, the meiocytes become encased in callose and a locular cavity forms. A rudimentary pollen wall, preceded by primexine deposition, is formed at the tetrad stage around the microspores before their release from the callose. The tapetum remains peripheral in the locule until the vacuolate pollen stage when it disappears. The sporogenous cells of the cytoplasmic male-sterile line complete meiosis, and the callose-encased microspores also deposit a primexine. Further development of the microspores is arrested. Before and during meiosis the tapetal cells become highly vacuolate and remain appressed to the meiocytes; a locular cavity is not formed. After primexine deposition, the tetrads of microspores, which are still encased in callose, seem to collapse as they are encroached upon by the vacuolate tapetum. After abortion of the microspores the outer tapetal layer degenerates, followed by the inner tapetal layer. The aborted mass late in anther development consists of crushed microspore tetrads, primary walls of the sporogenous cells and tapetum, callose, and the collapsed tapetum. The manner of abortion in pepper is compared with previously described mechanisms.