Rice Putative Pectin Methyltransferase Gene OsPMT10 Is Required for Maintaining the Cell Wall Properties of Pistil Transmitting Tissues via Pectin Modification.

Precise directional control of pollen tube growth via mechanical guidance by pistil tissue is critical for the successful fertilization of flowering plants and requires active cell-to-cell communication and maintenance of softness in the transmitting tissue. However, the regulation of transmitting tissue softness as controlled by cell wall properties, especially pectin, has not been reported. Here we report that regulation of pectin methylesterification supports pollen elongation through pistil transmitting tissues in Oryza sativa. The rice pectin methylesterase gene OsPMT10 was strongly expressed in reproductive tissues, especially the pistil. The ospmt10 mutant did not have a significant effect on vegetative growth, but the fertility rate was reduced by approximately half. In the ospmt10 mutant, pollen tube elongation was observed in the transmitting tissue of the style, but approximately half of the pollen tubes did not extend all the way to the ovule. Tissue cross-sections of the upper ovary were prepared, and immunohistochemical staining using LM19 and LM20 showed that methylesterified pectin distribution was decreased in ospmt10 compared with the wild type. The decreased expression of methylesterified pectins in ospmt10 may have resulted in loss of fluidity in the apoplast space of the transmitting tissue, rendering it difficult for the pollen tube to elongate in the transmitting tissue and thereby preventing it from reaching the ovule.

Paving the Way for Fertilization: The Role of the Transmitting Tract.

Angiosperm reproduction relies on the precise growth of the pollen tube through different pistil tissues carrying two sperm cells into the ovules' embryo sac, where they fuse with the egg and the central cell to accomplish double fertilization and ultimately initiate seed development. A network of intrinsic and tightly regulated communication and signaling cascades, which mediate continuous interactions between the pollen tube and the sporophytic and gametophytic female tissues, ensures the fast and meticulous growth of pollen tubes along the pistil, until it reaches the ovule embryo sac. Most of the pollen tube growth occurs in a specialized tissue-the transmitting tract-connecting the stigma, the style, and the ovary. This tissue is composed of highly secretory cells responsible for producing an extensive extracellular matrix. This multifaceted matrix is proposed to support and provide nutrition and adhesion for pollen tube growth and guidance. Insights pertaining to the mechanisms that underlie these processes remain sparse due to the difficulty of accessing and manipulating the female sporophytic tissues enclosed in the pistil. Here, we summarize the current knowledge on this key step of reproduction in flowering plants with special emphasis on the female transmitting tract tissue.

Contrasting models of the female reproductive tract in four o'clocks (Nyctaginaceae).

UNLABELLED: • PREMISE OF THE STUDY: In angiosperms, several carpel tissues are specialized to facilitate pollen-tube elongation to achieve fertilization. We evaluated the possible evolutionary pathways of the diverse female reproductive tracts in Nyctaginaceae.• METHODS: We studied the anatomy of a range of species representing different tribes, using light, fluorescence, scanning electron, and transmission electron microscopy.• KEY RESULTS: Stigmas have multicellular, multiseriate papillae, except for Boerhavia diffusa with unicellular papillae. The styles are solid, with a strand of transmitting tissue linking the stigma with the ventral ovary wall. In Allionia, Boerhavia, and Mirabilis, the transmitting tissue branches into two independent tracts at the base of the ovary and continues across the lateral margins of the funicle to the micropyle; it is composed of cells with thick walls surrounded by abundant extracellular matrix. Bougainvillea, Pisonia, and Pisoniella have a diffuse transmitting tissue and an obturator, a proliferation of cells covered by a layer of secretory papillae that encloses the funicle, placenta, and ventral wall of the gynoecium and contacts with the micropyle.• CONCLUSIONS: We propose two models of female reproductive tract, (A) one in which an obturator is absent and the transmitting tissue is compact and branched and (B) one in which an obturator is present and the transmitting tissue is diffuse. On the basis of character optimization, we hypothesize that model B represents the ancestral (plesiomorphic) condition in the family and model A originated once during evolution, within the tribe Nyctagineae.

SPL8 and miR156-targeted SPL genes redundantly regulate Arabidopsis gynoecium differential patterning.

SPL8 and miR156-targeted SPL genes are known to play an essential role in Arabidopsis anther development. Here we show that these SPL genes are also expressed within the developing gynoecium, where they redundantly control development of the female reproductive tract. Whereas the gynoecium morphology in the spl8 single mutant is largely normal, additional down-regulation of miR156-targeted SPL genes results in a shortened style and an apically swollen ovary narrowing onto an elongated gynophore. In particular, the septum does not form properly and lacks a transmitting tract. Loss of SPL8 function enhances the mutant phenotypes of ett, crc and spt, indicating a functional overlap between SPL8 and these genes in regulating gynoecium development. Furthermore, gynoecium development of 35S:MIR156b spl8-1 double mutants shows enhanced sensitivity to a polar auxin transport inhibitor, and the expression pattern of the auxin biosynthesis gene YUCCA4 is altered compared to wild-type. Our observations imply that SPL8 and miR156-targeted SPL genes control gynoecium patterning through interference with auxin homeostasis and signalling.

Rice Putative Methyltransferase Gene OsPMT16 Is Required for Pistil Development Involving Pectin Modification.

Pectin synthesis and modification are vital for plant development, although the underlying mechanisms are still not well understood. Furthermore, reports on the function of pectin in the pistil are limited. Herein, we report the functional characterization of the OsPMT16 gene, which encodes a putative pectin methyltransferase (PMT) in rice. The cell walls of rice leaves contain less pectin, and chemical analysis of pectin in the flower organ had not been previously performed. Therefore, in the present study, the amount of pectin in the reproductive tissues of rice was investigated. Of the reproductive tissues, the pistil was especially rich in pectin; thus, we focused on the pistil. OsPMT16 expression was confirmed in the pistil, and effects of pectin methylesterification regulation on the reproductive stage were investigated by studying the phenotype of the T-DNA insertion mutant. The ospmt16 mutant showed significantly reduced fertility. When the flowers were observed, tissue morphogenesis was abnormal in the pistil. Immunofluorescence staining by pectin-specific monoclonal antibodies of the pistil revealed that total pectin and esterified pectin were decreased among ospmt16 mutants. These results indicate that OsPMT16 contributes significantly to pistil development during reproductive growth.

Nicotiana tabacum pollen-pistil interactions show unexpected spatial and temporal differences in pollen tube growth among genotypes.

KEY MESSAGE: This research revealed diverse PTG rates among intraspecific pollen-pistil interactions that showed variable dependency on the stigma and mature TT. Pollen-pistil interactions regulate pollen tube growth (PTG) rates and are determinants of fertilization and seed set. This research focuses on the diversity of intraspecific PTG rates and the spatial and temporal regulation of PTG among Nicotiana tabacum genotypes. Nonrandom mating within self-compatible species has been noted, but little is known on the mechanisms involved. To begin research on nonrandom mating, we took advantage of the model reproductive system of N. tabacum and used seventeen diverse N. tabacum genotypes in a complete pollination diallel to measure the diversity of intraspecific pollen-pistil interactions. The 289 intraspecific interactions showed surprisingly large differences in PTG rates. The interaction between specific males and females resulted in 18 specific combining abilities that were significantly different, indicating the importance of the specific genotype interaction in regulating intraspecific PTG. No single female or male genotype exerted overall control of PTG rates, as determined by a general combining ability analysis. Slow and fast pollen-pistil interactions showed spatial differences in growth rates along the style. Slower interactions had a slower initial PTG rate while fast interactions had faster consistent rates of growth indicating spatial regulation of PTG in the pistil. Removal of the stigma or the mature transmitting tissue (TT) showed the tissue-specific component of PTG regulation. Stigma removal resulted in slower or no change in PTG rate depending on the pollen and pistil genotypes. Removal of the TT, which necessitated removal of the stigma, showed no change, slower or unexpectedly, increased growth rates relative to growth rates without a stigma. These data show the diverse nature of pollen-pistil interactions in N. tabacum genotypes providing a system to further investigate the regulation of PTG.

Calreticulin localizes to plant intra/extracellular peripheries of highly specialized cells involved in pollen-pistil interactions.

Calcium (Ca2+) plays essential roles in generative reproduction of angiosperms, but the sites and mechanisms of Ca2+ storage and mobilization during pollen-pistil interactions have not been fully defined. Both external and internal Ca2+ stores are likely important during male gametophyte communication with the sporophytic and gametophytic cells within the pistil. Given that calreticulin (CRT), a Ca2+-buffering protein, is able to bind Ca2+ reversibly, it can serve as a mobile store of easily releasable Ca2+ (so called an exchangeable Ca2+) in eukaryotic cells. CRT has typical endoplasmic reticulum (ER) targeting and retention signals and resides primarily in the ER. However, localization of this protein outside the ER has also been revealed in both animal and plant cells, including Golgi/dictyosomes, nucleus, plasma membrane/cell surface, plasmodesmata, and even extracellular matrix. These findings indicate that CRT may function in a variety of different cell compartments and specialized structures. We have recently shown that CRT is highly expressed and accumulated in the ER of plant cells involved in pollen-pistil interactions in Petunia, and we proposed an essential role for CRT in intracellular Ca2+ storage and mobilization during the key reproductive events. Here, we demonstrate that both CRT and exchangeable Ca2+ are localized in the intra/extracellular peripheries of highly specialized plant cells, such as the pistil transmitting tract cells, pollen tubes, nucellus cells surrounding the embryo sac, and synergids. Based on our present results, we propose that extracellularly located CRT is also involved in Ca2+ storage and mobilization during sexual reproduction of angiosperms.

Style morphology and pollen tube pathway.

The style morphology and anatomy vary among different species. Three basic types are: open, closed, and semi-closed. Cells involved in the pollen tube pathway in the different types of styles present abundant endoplasmic reticulum, dictyosomes, mitochondria, and ribosomes. These secretory characteristics are related to the secretion where pollen tube grows. This secretion can be represented by the substances either in the canal or in the intercellular matrix or in the cell wall. Most studies suggest that pollen tubes only grow through the secretion of the canal in open styles. However, some species present pollen tubes that penetrate the epithelial cells of the canal, or grow through the middle lamella between these cells and subepithelial cells. In species with a closed style, a pathway is provided by the presence of an extracellular matrix, or by the thickened cell walls of the stylar transmitting tissue. There are reports in some species where pollen tubes can also penetrate the transmitting tissue cells and continue their growth through the cell lumen. In this review, we define subtypes of styles according to the path of the pollen tube. Style types were mapped on an angiosperm phylogenetic tree following the maximum parsimony principle. In line with this, it could be hypothesized that: the open style appeared in the early divergent angiosperms; the closed type of style originated in Asparagales, Poales, and Eudicots; and the semi-closed style appeared in Rosids, Ericales, and Gentianales. The open style seems to have been lost in core Eudicots, with reversions in some Rosids and Asterids.

Immunodetection of some pectic, arabinogalactan proteins and hemicellulose epitopes in the micropylar transmitting tissue of apomictic dandelions (Taraxacum, Asteraceae, Lactuceae).

In apomictic Taraxacum species, the development of both the embryo and the endosperm does not require double fertilisation. However, a structural reduction of ovular transmitting tissue was not observed in apomictic dandelions. The aim of this study was to analyse the chemical composition of the cell walls to describe the presence of arabinogalactan proteins (AGPs), hemicellulose and some pectic epitopes in the micropylar transmitting tissue of apomictic Taraxacum. The results point to (1) the similar distribution of AGPs in different developmental stages, (2) the absence of highly methyl-esterified homogalacturonan (HG) in transmitting tissue of ovule containing a mature embryo sac and the appearance of this pectin domain in the young seed containing the embryo and endosperm, (3) the similar pattern of low methyl-esterified pectin occurrence in both an ovule and a young seed with an embryo and endosperm in apomictic Taraxacum and (4) the presence of hemicelluloses recognised by LM25 and LM21 antibodies in the reproductive structure of Taraxacum.

SF21 is a Protein which Exhibits a Dual Nuclear and Cytoplasmic Localization in Developing Pistils of Sunflower and Tobacco.

SF21 was originally described as a pollen- and pistil-expressed protein from sunflower and tobacco. In pistils excised from these species, transcripts were detected in the stigma and in the transmitting tissue where they accumulated in an ovary-oriented increasing concentration gradient. We studied the cellular localization of the SF21 protein during various stages of pistil development as well as in pollen grains of tobacco and sunflower. Here we demonstrate that in young tobacco pistils (from stage 2 onwards) this protein is expressed exclusively in the papillae and secretory cells of the stigma where it is located first in the nucleus and subsequently also in the cytoplasm. Only several stages later (stage 10) does it appear in the transmitting tissue cells of the style where it exhibits a similar, but temporally-delayed, dual nuclear and cytoplasmic localization pattern. SF21 is no longer present in either the stigma or style at the time of pollination, indicating that it is not directly involved in the pollination process. In tobacco pollen grains, SF21 appears just prior to pollen germination and localizes to the apical region of growing pollen tubes, suggesting a possible role in pollen tube growth. This temporal and spatial expression pattern as well as the dual nuclear/cytoplasmic localization suggest that SF21 could be involved in several molecular functions during pistil development and pollen tube growth.

Characterization of PhPRP1, a histidine domain arabinogalactan protein from Petunia hybrida pistils.

An arabinogalactan protein, PhPRP1, was purified from Petunia hybrida pistils and shown to be orthologous to TTS-1 and TTS-2 from Nicotiana tabacum and NaTTS from Nicotiana alata. Sequence comparisons among these proteins, and CaPRP1 from Capsicum annuum, reveal a conserved histidine-rich domain and two hypervariable domains. Immunoblots show that TTS-1 and PhPRP1 are also expressed in vegetative tissues of tobacco and petunia respectively. In contrast to the molecular mass heterogeneity displayed by the pistil proteins, the different isoforms found in seedlings, roots, and leaves each has a discrete size (37, 80, 160, and 200 kDa) on SDS-PAGE gels. On the basis of their chemistry, distinctive domain architecture, and the unique pattern of expression, we have named this group of proteins HD-AGPs (histidine domain-arabinogalactan proteins).