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.     

Disruption of the novel plant protein NEF1 affects lipid accumulation in the plastids of the tapetum and exine formation of pollen, resulting in male sterility in Arabidopsis thaliana

A novel male-sterile mutant of Arabidopsis thaliana was isolated by means of T-DNA tagging. Pollen abortion of the mutant was evident after microspore release, and pollen grains were completely absent at anthesis. Transmission electron microscope analysis revealed that primexine was coarsely developed, and that although sporopollenin was produced, it was not deposited onto the microspore plasma membrane. The sporopollenin that failed to be deposited aggregated and accumulated within the locule and on the locule wall. Finally, as no exine formation was observed, the mutant was named nef1. The plastoglobuli within the plastids of the tapetum were reduced, and lipid accumulation was considerably decreased. The mutant had a significantly altered leaf chloroplast ultrastructure and showed various growth defects. Lipid analysis revealed that the total lipid content in nef1 was lower than that in the wild type, which indicated that Nef1 was involved in lipid metabolism. Cloning of the full-length Nef1 indicated that the gene encodes a novel plant protein of 1123 amino acids with limited sequence similarities to membrane proteins or transporter-like proteins, and the NEF1 is predicted to be a plastid integral membrane protein. Motif analysis revealed that NEF1 contains prokaryotic membrane lipoprotein lipid attachment sites that are involved in maintaining cell envelope integrity. It is predicted that the Nef1 encodes a membrane protein that maintains the envelope integrity in the plastids.     

A comparative ultrastructural analysis of exine pattern development in wild-typeArabidopsis and a mutant defective in pattern formation

Summary In order to identify factors necessary for the establishment of the reticulate pollen wall pattern, we have characterized a T-DNA tagged mutant ofArabidopsis thaliana that is defective in pattern formation. This study reports the results of an ultrastructural comparison of pollen wall formation in the mutant to wall development in wild-type plants. Pollen wall development in the mutant parallels that of wild-type until the early tetrad stage. At this point in wild-type plants, the microspore plasma membrane assumes a regular pattern of ridges and valleys. Initial sporopollenin deposition occurs on the ridges marking the beginning of probacula formation. In contrast, the plasma membrane in the mutant appears irregular with flattened protuberances and rare invaginations. As a result, the wild-type regular pattern of ridges and valleys is not formed. Sporopollenin is randomly deposited on the plasma membrane and aggregates on the locule wall; it is not anchored to the membrane. Our finding that the mutation blocks the normal invagination of the plasma membrane and disrupts the proper deposition of sporopollenin during wall formation suggests that the mutation could be in a gene responsible for pattern formation. These results also provide direct evidence that the plasma membrane plays a critical role in the establishment of the pollen wall pattern.     

The HKM gene, which is identical to the MS1 gene of Arabidopsis thaliana, is essential for primexine formation and exine pattern formation

A male-sterile mutant of Arabidopsis thaliana was isolated by T-DNA tagging screening. Using transmission electron microscopy analysis, we revealed that the microspores of this mutant did not have normal thick primexine on the microspore at the tetrad stage. Instead, a moderately electron-dense layer formed around the microspores. Although microspores without normal primexine failed to form a proper reticulate exine pattern at later stages, sporopollenin was deposited and an exine-like hackly structure was observed on the microspores during the microspore stage. Thus, this mutant was named hackly microspore (hkm). It is speculated that the moderately electron-dense layer was primexine, which partially played its role in sporopollenin deposition onto the microspore. Cytological analysis revealed that the tapetum of the hkm mutant was significantly vacuolated, and that vacuolated tapetal cells crushed the microspores, resulting in the absence of pollen grains within the anther at anthesis. Single nucleotide polymorphism analysis demonstrated that the hkm mutation exists within the MS1 gene, which has been reportedly expressed within the tapetum. Our results suggest that the critical process of primexine formation is under sporophytic control .     

莴苣花药发育过程中钙的分布特征

减数分裂前,莴苣花药中的钙颗粒很少。减数分裂后,花药绒毡层细胞中的钙颗粒明显增加。同时在花药药室基质中也出现许多细小的钙颗粒。刚从四分体中释放出的小孢子内钙颗粒很少。伴随着花粉外壁物质在小孢子表面的沉积,钙颗粒开始积累在花粉壁部位。随后。小孢子中开始出现钙颗粒。当小孢子开始形成液泡后,钙颗粒向其中聚集,伴随着小液泡融合成大液泡。体积较大的钙颗粒主要集中在液泡中,而细胞质基质中的钙颗粒很少。随着二胞花粉中的大液泡消失,花粉细胞质中的钙颗粒变得很少。在以后的发育中,只有花粉壁中积累较多的钙颗粒。在莴苣花药发育过程中,钙与绒毡层细胞的退化和小孢子液泡形成以及二胞花粉中大液泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。     

莴苣花药发育过程中钙的分布特征

减数分裂前,莴苣花药中的钙颗粒很少。减数分裂后,花药绒毡层细胞中的钙颗粒明显增加, 同时在花药药室基质中也出现许多细小的钙颗粒。刚从四分体中释放出的小孢子内钙颗粒很少,伴随着花粉外壁物质在小孢子表面的沉积,钙颗粒开始积累在花粉壁部位。随后,小孢子中开始出现钙颗粒。当小孢子开始形成液泡后,钙颗粒向其中聚集,伴随着小液泡融合成大液泡,体积较大的钙颗粒主要集中在液泡中,而细胞质基质中的钙颗粒很少。随着二胞花粉中的大液泡消失,花粉细胞质中的钙颗粒变得很少。在以后的发育中,只有花粉壁中积累较多的钙颗粒。在莴苣花药发育过程中,钙与绒毡层细胞的退化和小孢子液泡形成以及二胞花粉中大波泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。     

A novel male-sterile mutant of Arabidopsis thaliana,faceless pollen-1, produces pollen with a smooth surface and an acetolysis-sensitive exine

A mutant exhibiting conditional male sterility, in which fertility was restored under conditions of high humidity, was identified in T-DNA tagged lines of Arabidopsis thaliana. Scanning electron microscopy (SEM) demonstrated that the pollen surface was almost smooth and the reticulate pattern not prominent. Thus, the mutant was named faceless pollen-1 (flp1). Transmission electron microscopy (TEM) revealed that the smooth appearance was due to tryphine filling in the exine cavities and covering the pollen surface. The lipid droplets in the tryphine of mutant pollen were smaller and more numerous than those of the wild type. SEM analysis also demonstrated that pollen exine was easily damaged by acetolysis, suggesting that a component of exine, sporopollenin, was defective in the mutant. In addition, the stems and siliques had reduced amounts of wax crystals. A predicted amino acid sequence of the cDNA that corresponded to the tagged gene, fip1, showed sequence similarity to proteins involved in wax biosynthesis. The FLP1 protein is likely to play a role in the synthesis of the components of tryphine, sporopollenin of exine and the wax of stems and siliques.     

Identification of kaonashi mutants showing abnormal pollen exine structure in Arabidopsis thaliana

Exine, the outermost architecture of pollen walls, protects male gametes from the environment by virtue of its chemical and physical stability. Although much effort has been devoted to revealing the mechanism of exine construction, still little is known about it. To identify the genes involved in exine formation, we screened for Arabidopsis mutants with pollen grains exhibiting abnormal exine structure using scanning electron microscopy. We isolated 12 mutants, kaonashi1 (kns1) to kns12, and classified them into four types. The type 1 mutants showed a collapsed exine structure resembling a mutant of the callose synthase gene, suggesting that the type 1 genes are involved in callose wall synthesis. The type 2 mutant showed remarkably thin exine structure, presumably due to defective primexine thickening. The type 3 mutants showed defective tectum formation, and thus type 3 genes are required for primordial tectum formation or biosynthesis and deposition of sporopollenin. The type 4 mutants showed densely distributed baculae, suggesting type 4 genes determine the position of probacula formation. All identified kns mutants were recessive, suggesting that these KNS genes are expressed in sporophytic cells. Unlike previously known exine-defective mutants, most of the kns mutants showed normal fertility. Map-based cloning revealed that KNS2, one of the type 4 genes, encodes sucrose phosphate synthase. This enzyme might be required for synthesis of primexine or callose wall, which are both important for probacula positioning. Analysis of kns mutants will provide new knowledge to help understand the mechanism of biosynthesis of exine components and the construction of exine architecture.     

A glycine-rich protein that facilitates exine formation during tomato pollen development

Formation of the unique and highly diverse outer cell wall, or exine, of pollen is essential for normal pollen function and survival. However, little is known about the many contributing proteins and processes involved in the formation of this wall. The tomato gene LeGRP92 encodes for a glycine-rich protein produced specifically in the tapetum. LeGRP92 is found as four major forms that accumulate differentially in protein extracts from stamens at different developmental stages. The three largest molecular weight forms accumulated during early microspore development, while the smallest molecular weight form of LeGRP92 was present in protein extracts from stamens from early microsporogenesis through anther dehiscence, and was the only form present in dehisced pollen. Light microscopy immunolocalization experiments detected LeGRP92 at only two stages, late tetrad and early free microspore. However, we observed accumulation of the LeGRP92 at the early tetrad stage of development by removing the callose wall from tetrads, which allowed LeGRP92 detection. Transmission electron microscopy confirmed the LeGRP92 accumulation from microspore mother cells, tetrads through anther dehiscence. It was observed in the callose surrounding the microspore mother cells and tetrads, the exine of microspores and mature pollen, and orbicules. Plants expressing antisense RNA had reduced levels of LeGRP92 mRNA and protein, which correlated to pollen with altered exine formation and reduced pollen viability and germination. These data suggest that the LeGRP92 has a role in facilitating sporopollenin deposition and uniform exine formation and pollen viability.     

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.     

Disruption of a gene encoding C4-dicarboxylate transporter-like protein increases ozone sensitivity through deregulation of the stomatal response in Arabidopsis thaliana

To understand better the plant response to ozone, we isolated and characterized an ozone-sensitive (ozs1) mutant strain from a set of T-DNA-tagged Arabidopsis thaliana ecotype Columbia. The mutant plants show enhanced sensitivity to ozone, desiccation and sulfur dioxide, but have normal sensitivity to hydrogen peroxide, low temperature and high light levels. The T-DNA was inserted at a single locus which is linked to ozone sensitivity. Identification of the genomic sequences flanking the T-DNA insertion revealed disruption of a gene encoding a transporter-like protein of the tellurite resistance/C(4)-dicarboxylate transporter family. Plants with either of two different T-DNA insertions in this gene were also sensitive to ozone, and these plants failed to complement ozs1. Transpiration levels, stomatal conductance levels and the size of stomatal apertures were greater in ozs1 mutant plants than in the wild type. The stomatal apertures of ozs1 mutant plants responded to light fluctuations but were always larger than those of the wild-type plants under the same conditions. The stomata of the mutant and wild-type plants responded similarly to stimuli such as light, abscisic acid, high concentrations of carbon dioxide and ozone. These results suggest that OZS1 helps to close stomata, being not involved in the responses to these signals.     

Chloroplast NAD kinase is essential for energy transduction through the xanthophyll cycle in photosynthesis

Photosynthetic parameters of the nadk2 mutant of Arabidopsis thaliana, which is defective in chloroplast NAD kinase, were investigated. In this plant, the effective efficiency of photosynthetic electron transport (PhiII) and the quantum yield of open reaction centers of photosystem II (Fv'/Fm') were decreased. Furthermore, an increase in non-photochemical quenching attributed to energy dissipation from the xanthophyll cycle was observed. The mutant showed an aberrant de-epoxidation state of xanthophyll cycle carotenoids and had a high level of zeaxanthin even under low light conditions. These results indicate that chloroplast NAD kinase, catalyzing phosphorylation of NAD, is essential for the proper photosynthetic machinery of PSII and the xanthophyll cycle.     

ABNORMAL POLLEN VACUOLATION1 (APV1) is required for male fertility by contributing to anther cuticle and pollen exine formation in maize

Anther cuticle and pollen exine are the major protective barriers against various stresses. The proper functioning of genes expressed in the tapetum is vital for the development of pollen exine and anther cuticle. In this study, we report a tapetum‐specific gene, Abnormal Pollen Vacuolation1 (APV1), in maize that affects anther cuticle and pollen exine formation. The apv1 mutant was completely male sterile. Its microspores were swollen, less vacuolated, with a flat and empty anther locule. In the mutant, the anther epidermal surface was smooth, shiny, and plate‐shaped compared with the three‐dimensional crowded ridges and randomly formed wax crystals on the epidermal surface of the wild‐type. The wild‐type mature pollen had elaborate exine patterning, whereas the apv1 pollen surface was smooth. Only a few unevenly distributed Ubisch bodies were formed on the apv1 mutant, leading to a more apparent inner surface. A significant reduction in the cutin monomers was observed in the mutant. APV1 encodes a member of the P450 subfamily, CYP703A2‐Zm, which contains 530 amino acids. APV1 appeared to be widely expressed in the tapetum at the vacuolation stage, and its protein signal co‐localized with the endoplasmic reticulum (ER) signal. RNA‐Seq data revealed that most of the genes in the fatty acid metabolism pathway were differentially expressed in the apv1 mutant. Altogether, we suggest that APV1 functions in the fatty acid hydroxylation pathway which is involved in forming sporopollenin precursors and cutin monomers that are essential for the development of pollen exine and anther cuticle in maize.     

Development of the pollen grain and tapetum of wheat (Triticum aestivum) in untreated plants and plants treated with chemical hybridizing agent RH0007

Summary A study of pollen development in wheat was made using transmission electron microscopy (TEM). Microspores contain undifferentiated plastids and mitochondria that are dividing. Vacuolation occurs, probably due to the coalescence of small vacuoles budded off the endoplasmic reticulum (ER). As the pollen grain is formed and matures, the ER becomes distended with deposits of granular storage material. Mitochondria proliferate and become filled with cristae. Similarly, plastids divide and accumulate starch. The exine wall is deposited at a rapid rate throughout development, and the precursors appear to be synthesized in the tapetum. Tapetal cells become binucleate during the meiosis stage, and Ubisch bodies form on the plasma membrane surface that faces the locule. Tapetal plastids become surrounded by an electron-translucent halo. Rough ER is associated with the halo around the plastids and with the plasma membrane. We hypothesize that the sporopollenin precursors for both the Ubisch bodies and exine pollen wall are synthesized in the tapetal plastids and are transported to the tapetal cell surface via the ER. The microspore plastids appear to be involved in activities other than precursor synthesis: plastid proliferation in young microspores, and starch synthesis later in development. Plants treated with the chemical hybridizing agent RH0007 show a pattern of development similar to that shown by untreated control plants through the meiosis stage. In the young microspore stage the exine wall is deposited irregularly and is thinner than that of control plants. In many cases the microspores are seen to have wavy contours. With the onset of vacuolation, microspores become plasmolyzed and abort. The tapetal cells in RH0007-treated locules divide normally through the meiosis stage. Less sporopollenin is deposited in the Ubisch bodies, and the pattern is less regular than that of the control. In many cases, the tapetal cells expand into the locule. At the base of one of the locules treated with a dosage of RH0007 that causes 95% male sterility, several microspores survived and developed into pollen grains that were sterile. The conditions at the base of the locule may have reduced the osmotic stress on the microspores, allowing them to survive. Preliminary work showed that the extractable quantity of carotenoids in RHOOO7-treated anthers was slightly greater than in controls. We concluded that RH0007 appears to interfere with the polymerization of carotenoid precursors into the exine wall and Ubisch bodies, rather than interfering with the synthesis of the precursors.     

New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana

Background and Aims

The Arabidopsis thaliana pollen cell wall is a complex structure consisting of an outer sporopollenin framework and lipid-rich coat, as well as an inner cellulosic wall. Although mutant analysis has been a useful tool to study pollen cell walls, the ultrastructure of the arabidopsis anther has proved to be challenging to preserve for electron microscopy.

Methods

In this work, high-pressure freezing/freeze substitution and transmission electron microscopy were used to examine the sequence of developmental events in the anther that lead to sporopollenin deposition to form the exine and the dramatic differentiation and death of the tapetum, which produces the pollen coat.

Key Results

Cryo-fixation revealed a new view of the interplay between sporophytic anther tissues and gametophytic microspores over the course of pollen development, especially with respect to the intact microspore/pollen wall and the continuous tapetum epithelium. These data reveal the ultrastructure of tapetosomes and elaioplasts, highly specialized tapetum organelles that accumulate pollen coat components. The tapetum and middle layer of the anther also remain intact into the tricellular pollen and late uninucleate microspore stages, respectively.

Conclusions

This high-quality structural information, interpreted in the context of recent functional studies, provides the groundwork for future mutant studies where tapetum and microspore ultrastructure is assessed.     

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.     

Anther structure and pollen development in Melicoccus lepidopetalus (Sapindaceae): An evolutionary approach to dioecy in the family

Anther and pollen development in staminate and pistillate flowers of dioecious Melicoccus lepidopetalus (Sapindaceae) were examined by light and electron microscopy. Young anthers are similar in both types of flowers; they consist of epidermis, endothecium, two to four middle layers and a secretory tapetum. The microspore tetrads are tetrahedral. The mature anther in staminate flowers presents compressed epidermal cells and endothecium cells with fibrillar thickenings. A single locule is formed in the theca by dissolution of the septum and pollen grains are shed at two-celled stage. The mature anthers of pistillate flowers differ anatomically from those of staminate flowers. The epidermis is not compressed, the endothecium does not develop fibrillar thickenings, middle layers and tapetum are generally persisting, and the stomium is nonfunctional. Microspore degeneration begins after meiosis of microspore mother cells. At anthesis, uninucleate microspores and pollen grains with vegetative and generative nuclei with no cytokinesis are observed. Some pollen walls display an abnormal exine deposition, whereas others show a well formed exine, although both are devoid of intine. These results suggest that in the evolution towards unisexuality, the developmental differences of anther wall tissues and pollen grains between pistillate and staminate flowers might become more pronounced in a derived condition, such as dioecy.