Differential localization of cell wall polymers across generations in the placenta of Marchantia polymorpha.

To further knowledge on cell wall composition in early land plants, we localized cell wall constituents in placental cells of the liverwort Marchantia polymorpha L. using monoclonal antibodies (MAbs) in the transmission electron microscope and histochemical staining. The placenta of M. polymorpha is similar to the majority of bryophytes in that both generations contain transfer cells with extensive wall ingrowths. Although the four major cell wall polymers, i.e., cellulose, pectins, hemicelluloses, and arabinogalactan proteins, are present, there are variations in the richness and specificity across generations. An abundance of homogalacturonan pectins in all placental cell walls is consistent with maintaining cell wall permeability and an acidic apoplastic pH necessary for solute transport. Although similar in ultrastructure, transfer cell walls on the sporophyte side in M. polymorpha are enriched with xyloglucans and diverse AGPs not detected on the gametophyte side of the placenta. Gametophyte wall ingrowths are more uniform in polymer composition. Lastly, extensins and callose are not components of transfer cell walls of M. polymorpha, which deviates from studies on transfer cells in other plants. The difference in polymer localizations in transfer cell walls between generations is consistent with directional movement from gametophyte to sporophyte in this liverwort.

Distinct Differentiation Characteristics of Endothelium Determine Its Ability to Form Pseudo-Embryos in Tomato Ovules.

The endothelium is an additional cell layer, differentiating from the inner epidermis of the ovule integument. In tomato (Solanum lycopersicum L.), after fertilization, the endothelium separates from integument and becomes an independent tissue developing next to the growing embryo sac. In the absence of fertilization, the endothelium may proliferate and form pseudo-embryo. However, the course of the reorganization of endothelium into pseudo-embryo in tomato ovules is poorly understood. We aimed to investigate specific features of endothelium differentiation and the role of the endothelium in the development of fertilized and unfertilized tomato ovules. The ovules of tomato plants ("YaLF" line), produced by vegetative growth plants of transgenic tomato line expressing the ac gene, encoding chitin-binding protein from Amaranthus caudatus L., were investigated using light and transmission electron microscopy. We showed that in the fertilized ovule of normally developing fruit and in the unfertilized ovule of parthenocarpic fruit, separation of the endothelium from integument occurs via programmed death of cells of the integumental parenchyma, adjacent to the endothelium. Endothelial cells in normally developing ovules change their structural and functional specialization from meristematic to secretory and back to meristematic, and proliferate until seeds fully mature. The secretory activity of the endothelium is necessary for the lysis of dying cells of the integument and provides the space for the growth of the new sporophyte. However, in ovules of parthenocarpic fruits, pseudo-embryo cells do not change their structural and functional organization and remain meristematic, no zone of lysis is formed, and pseudo-embryo cells undergo programmed cell death. Our data shows the key role of the endothelium as a protective and secretory tissue, needed for the normal development of ovules.

ARF2-ARF4 and ARF5 are Essential for Female and Male Gametophyte Development in Arabidopsis.

The plant hormone auxin plays critical roles in plant growth and development. Auxin response factors (ARFs) are a class of transcription factors which regulate auxin-mediated gene expression. While the functions of ARFs in sporophytic development have been well characterized, their functions specific to gametophytic development have not been studied extensively. In this study, Arabidopsis ARF genes were selectively down-regulated in gametophytes by misexpression of targeted microRNAs (amiRARF234, amiRARFMP and MIR167a) to silence AtARF2-AtAEF4, AtARF5, AtARF6 and AtARF8. Embryo sacs in amiRARF234- and amiRARFMP-expressing plants exhibited identity defects in cells at the micropylar pole, such as formation of two cells with egg cell-like morphology, concomitant with loss of synergid marker expression and seed abortion. The pollen grains of the transgenic plants were morphologically aberrant and unviable, and the inclusions and nuclei were lost in the abnormal pollen grains. However, plants misexpressing MIR167a showed no obvious abnormal phenotypes in the embryo sacs and pollen grains. Overall, these results provide evidence that AtARF2-AtARF4 and AtARF5 play significant roles in regulating both female and male gametophyte development in Arabidopsis.

The Ceratopteris (fern) developing motile gamete walls contain diverse polysaccharides, but not pectin.

MAIN CONCLUSION: Unlike most plant cell walls, the five consecutive walls laid down during spermatogenesis in the model fern Ceratopteris contain sparse cellulose, lack pectin and are enriched with callose and hemicelluloses. Seed-free plants like bryophytes and pteridophytes produce swimming male gametes for sexual reproduction. During spermatogenesis, unique walls are formed that are essential to the appropriate development and maturation of the motile gametes. Other than the detection of callose and general wall polysaccharides in scattered groups, little is known about the sequence of wall formation and the composition of these walls during sperm cell differentiation in plants that produce swimming sperm. Using histochemistry and immunogold localizations, we examined the distribution of callose, cellulose, mannan and xylan-containing hemicelluloses, and homogalacturonan (HG) pectins in the special walls deposited during spermatogenesis in Ceratopteris. Five walls are produced in sequence and each has a unique fate. The first wall (W1) contains callose and sparse xylan-containing hemicelluloses. Wall two (W2) is thin and composed of cellulose crosslinked by xylan-containing hemicelluloses. The third wall (W3) is thick and composed entirely of callose, and the fourth wall (W4) is built of cellulose heavily crosslinked by galactoxyloglucan hemicelluloses. Wall five (W5) is an arabinogalactan protein (AGP)-rich matrix in which the gamete changes shape and multiple flagella elongate. We detected no esterified or unesterified HG pectins in any of the walls laid down during spermatogenesis. To consider evolutionary modifications in cell walls associated with motile gametes, comparisons are presented with male gametophyte and spermatogenous cell walls across plant groups.

Grimmiaceae in the Early Cretaceous: Tricarinella crassiphylla gen. et sp. nov. and the value of anatomically preserved bryophytes.

Background and Aims: Widespread and diverse in modern ecosystems, mosses are rare in the fossil record, especially in pre-Cenozoic rocks. Furthermore, most pre-Cenozoic mosses are known from compression fossils, which lack detailed anatomical information. When preserved, anatomy significantly improves resolution in the systematic placement of fossils. Lower Cretaceous (Valanginian) deposits on Vancouver Island (British Columbia, Canada) contain a diverse anatomically preserved flora including numerous bryophytes, many of which have yet to be characterized. Among them is the grimmiaceous moss described here. Methods: One fossil moss gametophyte preserved in a carbonate concretion was studied in serial sections prepared using the cellulose acetate peel technique. Key Results: Tricarinella crassiphylla gen. et sp. nov. is a moss with tristichous phyllotaxis and strongly keeled leaves. The combination of an acrocarpous condition (inferred based on a series of morphological features), a central conducting strand, a homogeneous leaf costa and a lamina with bistratose portions and sinuous cells, and multicellular gemmae, supports placement of Tricarinella in family Grimmiaceae. Tricarinella is similar to Grimmia, a genus that exhibits broad morphological variability. However, tristichous phyllotaxis and especially the lamina, bistratose at the base but not in distal portions of the leaf, set Tricarinella apart as a distinct genus. Conclusions: Tricarinella crassiphylla marks the oldest record for both family Grimmiaceae and sub-class Dicranidae, providing a hard minimum age (136 million years) for these groups. The fact that this fossil could be placed in an extant family, despite a diminutive size, emphasizes the considerable resolving power of anatomically preserved bryophyte fossils, even when recovered from allochthonous assemblages of marine sediments, such as the Apple Bay flora. Discovery of Tricarinella re-emphasizes the importance of paleobotanical studies as the only approach allowing access to a significant segment of biodiversity, the extinct biodiversity, which is unattainable by other means of investigation.

The parthenocarpic hydra mutant reveals a new function for a SPOROCYTELESS-like gene in the control of fruit set in tomato.

Fruit set is an essential process to ensure successful sexual plant reproduction. The development of the flower into a fruit is actively repressed in the absence of pollination. However, some cultivars from a few species are able to develop seedless fruits overcoming the standard restriction of unpollinated ovaries to growth. We report here the identification of the tomato hydra mutant that produces seedless (parthenocarpic) fruits. Seedless fruit production in hydra plants is linked to the absence of both male and female sporocyte development. The HYDRA gene is therefore essential for the initiation of sporogenesis in tomato. Using positional cloning, virus-induced gene silencing and expression analysis experiments, we identified the HYDRA gene and demonstrated that it encodes the tomato orthologue of SPOROCYTELESS/NOZZLE (SPL/NZZ) of Arabidopsis. We found that the precocious growth of the ovary is associated with changes in the expression of genes involved in gibberellin (GA) metabolism. Our results support the conservation of the function of SPL-like genes in the control of sporogenesis in plants. Moreover, this study uncovers a new function for the tomato SlSPL/HYDRA gene in the control of fruit initiation.

Extending the fossil record of Polytrichaceae: Early Cretaceous Meantoinea alophosioides gen. et sp. nov., permineralized gametophytes with gemma cups from Vancouver Island.

PREMISE OF THE STUDY: Diverse in modern ecosystems, mosses are dramatically underrepresented in the fossil record. Furthermore, most pre-Cenozoic mosses are known only from compression fossils, lacking detailed anatomical information. When preserved, anatomy vastly improves resolution in the systematic placement of fossils. Lower Cretaceous deposits at Apple Bay (Vancouver Island, British Columbia, Canada) contain a diverse anatomically preserved flora that includes numerous bryophytes, many of which have yet to be characterized. Among them is a polytrichaceous moss that is described here. METHODS: Fossil moss gametophytes preserved in four carbonate concretions were studied in serial sections prepared using the cellulose acetate peel technique. KEY RESULTS: We describe Meantoinea alophosioides gen. et sp. nov., a polytrichaceous moss with terminal gemma cups containing stalked, lenticular gemmae. Leaves with characteristic costal anatomy, differentiated into sheathing base and free lamina and bearing photosynthetic lamellae, along with a conducting strand in the stem, place Meantoinea in family Polytrichaceae. The bistratose leaf lamina with an adaxial layer of mamillose cells, short photosynthetic lamellae restricted to the costa, and presence of gemma cups indicate affinities with basal members of the Polytrichaceae, such as Lyellia, Bartramiopsis, and Alophosia. CONCLUSIONS: Meantoinea alophosioides enriches the documented moss diversity of an already-diverse Early Cretaceous plant fossil assemblage. This is the third moss described from the Apple Bay plant fossil assemblage and represents the first occurrence of gemma cups in a fossil moss. It is also the oldest unequivocal record of Polytrichaceae, providing a hard minimum age for the group of 136 million years.

Fluorescent whole-mount RNA in situ hybridization (F-WISH) in plant germ cells and the fertilized ovule.

First evidence on gene function and regulation is provided by the cellular expression pattern in complex tissues. However, to understand the activity of a specific gene, it is essential to analyze the regulatory network, which controls the spatio-temporal translation pattern during the entire life span of the transcribed mRNA. To explore mechanisms which control mRNA abundance and localization in space and time, it is necessary to visualize mRNAs quantitatively with a subcellular resolution, without sectioning the tissues. We have adapted and optimized a protocol for colorimetric whole-mount RNA in situ hybridization (WISH) using egg cell-specific digoxigenin (DIG) labeled probes (Hejátko et al., 2006) [1] on ovules and early seeds of Arabidopsis. Furthermore, we established a fluorescent whole-mount RNA in situ hybridization (F-WISH) protocol, which allows mRNA visualization on a subcellular level. The polar localized mRNA of SBT4.13, encoding a subtilase, was identified using this protocol. Both methods are described and discussed in detail. Additionally a (F)-WISH flow-chart is provided along with a troubleshooting table.

The phenotype of the CRINKLY4 deletion mutant of Physcomitrella patens suggests a broad role in developmental regulation in early land plants.

MAIN CONCLUSIONS: Deletion of the ancestral gene of the land plant multigene family of receptor like kinase CR4 in Physcomitrella patens demonstrates involvement in developmental control of gametophytic and sporophytic organs. The CRINKLY4 (CR4) family of receptor kinases in angiosperms consists of three clades, one including CR4, the CR4-related CCR1 and CCR2, a second including CCR3 and CCR4 family members, and a third and more distant clade. In addition to crinkly leaves in maize, which gave rise to the mutant gene name, CR4 is implicated in ovule, embryo, flower and root development in Arabidopsis thaliana. In root tips of the same species the module including a CLAVATA3/ESR-related protein, an Arabidopsis CR4, a CLAVATA1 and a WUSCHEL-related homeobox 5 (CLE40-ACR4-CLV1-WOX5) is implicated in meristem cell regulation. In embryos and shoots, CR4 acts together with A. thaliana MERISTEM LAYER 1 and PROTODERMAL FACTOR 2 to promote A. thaliana epidermis differentiation. Phylogenetic analysis has demonstrated that early land plants, e.g. mosses carry a single ancestral CR4 gene, together with genes encoding the other members of the CLE40-ACR4-CLV1-WOX5 signaling module. Here we show that CR4 serves as a broad regulator of morphogenesis both in gametophyte phyllids, archegonia and in sporophyte epidermis of the moss Physcomitrella patens. The phenotype of the CR4 deletion mutant in moss provides insight into the role of the ancestral CR4 gene as a regulator of development in early land plants.

Spatiotemporal features of microsporogenesis in the cycad species Macrozamia communis.

UNLABELLED: • PREMISE OF THE STUDY: Spatiotemporal features of microsporogenesis may provide important clues about the evolution of microsporogenesis in seed plants. One cellular feature that attracts special attention is advance cell wall ingrowths (ACWIs) at future cytokinetic sites in microsporocytes since they have been found only in species of an ancient lineage of angiosperms, Magnolia, and in much less detail, of an ancient lineage of gymnosperms, cycads. Further investigation into microsporogenesis in a cycad species may yield knowledge critical to understanding the establishment of ACWIs as an important feature for comparative studies of microsporogenesis in seed plants.• METHODS: Bright-field and epifluorescence microscopy, confocal laser scanning microscopy, and transmission electron microscopy were used to investigate the microsporogenic process in Macrozamia communis, a species in the Zamiaceae family of cycads.• KEY RESULTS: In prophase-II microsporocytes in M. communis, ACWIs form as a callose ring between the newly formed nuclei and are not accompanied by cytokinetic apparatuses such as mini-phragmoplasts, wide tubules, or wide tubular networks. Shortly after the second nuclear division, new ACWIs, albeit thinner than the previous ACWIs, form between the newly formed nuclei. Subsequent cell plate formation in the planes of the ACWIs typically results in tetragonal tetrads.• CONCLUSIONS: Cytokinesis at the cell periphery is initiated earlier than cell plate formation in the cell interior in microsporogenesis in M. communis. The cellular features uncovered in M. communis may serve as useful reference features for comparative studies of microsporogenesis in plants.

Embryology of Phyllonoma (Phyllonomaceae, Aquifoliales): characteristics and character evolution.

Phyllonoma, the sole genus of Phyllonomaceae (Aquifoliales) consisting of four Central American species, has not been well-characterized morphologically. Following a previous study of flower and inflorescence morphology, I here report the embryology of the genus based on P. tenuidens and compare its characteristics with those of other aquifolialean families, namely, Aquifoliaceae, Cardiopteridaceae, Helwingiaceae, and Stemonuraceae. Comparisons indicate that although Phyllonoma resembles all the other families embryologically, it more closely resembles Aquifoliaceae and Helwingiaceae in lacking a vascular bundle in its integument and bearing ab initio Cellular endosperm. The genus especially resembles Helwingiaceae by possessing a tenuinucellate ovule. This result corroborates molecular and floral morphological evidence, supporting the distinctness of Phyllonoma as a family and its sister-group relationship to East‒Asian Helwingiaceae. However, Phyllonoma is clearly distinguished from Helwingiaceae by seed coat structure. In Phyllonoma, the seeds (dispersed in berries) have a thick seed coat composed of irregularly enlarged, thick-walled exotestal cells, whereas the seeds (dispersed in drupes) have a thin membranous seed coat in Helwingiaceae. Taken together with earlier information on pollination (entomophily in Phyllonoma versus ambophily in Helwingiaceae), embryological evidence shows that distinct evolution has occurred in reproductive characters relating to pollination and seed dispersal in Phyllonoma.

A Cd/Fe/Zn-responsive phytochelatin synthase is constitutively present in the ancient liverwort Lunularia cruciata (L.) dumort.

Lunularia cruciata occupies a very basal position in the phylogenetic tree of liverworts, which in turn have been recognized as a very early clade of land plants. It would therefore seem appropriate to take L. cruciata as the startingpoint for investigating character evolution in plants' metal(loid) response. One of the strongest evolutionary pressures for land colonization by plants has come from potential access to much greater amounts of nutritive ions from surface rocks, compared to water. This might have resulted in the need to precisely regulate trace element homeostasis and to minimize the risk of exposure to toxic concentrations of certain metals, prompting the evolution of a number of response mechanisms, such as synthesis of phytochelatins, metal(loid)-binding thiol-peptides. Accordingly, if the ability to synthesize phytochelatins and the occurrence of an active phytochelatin synthase are traits present in a basal liverwort species, and have been even reinforced in 'modern' tracheophytes, e.g. Arabidopsis thaliana, then such traits would presumably have played an essential role in plant fitness over time. Hence, we demonstrated here that: (i) L. cruciata compartmentalizes cadmium in the vacuoles of the phototosynthetic parenchyma by means of a phytochelatin-mediated detoxification strategy, and possesses a phytochelatin synthase that is activated by cadmium and homeostatic concentrations of iron(II) and zinc; and (ii) A. thaliana phytochelatin synthase displays a higher and broader response to several metal(loid)s [namely: cadmium, iron(II), zinc, copper, mercury, lead, arsenic(III)] than L. cruciata phytochelatin synthase.

Deficiency in a very-long-chain fatty acid ß-ketoacyl-coenzyme a synthase of tomato impairs microgametogenesis and causes floral organ fusion.

Previously, it was shown that ß-ketoacyl-coenzyme A synthase ECERIFERUM6 (CER6) is necessary for the biosynthesis of very-long-chain fatty acids with chain lengths beyond C28 in tomato (Solanum lycopersicum) fruits and C26 in Arabidopsis (Arabidopsis thaliana) leaves and the pollen coat. CER6 loss of function in Arabidopsis resulted in conditional male sterility, since pollen coat lipids are responsible for contact-mediated pollen hydration. In tomato, on the contrary, pollen hydration does not rely on pollen coat lipids. Nevertheless, mutation in SlCER6 impairs fertility and floral morphology. Here, the contribution of SlCER6 to the sexual reproduction and flower development of tomato was addressed. Cytological analysis and cross-pollination experiments revealed that the slcer6 mutant has male sterility caused by (1) hampered pollen dispersal and (2) abnormal tapetum development. SlCER6 loss of function provokes a decrease of n- and iso-alkanes with chain lengths of C27 or greater and of anteiso-alkanes with chain lengths of C28 or greater in flower cuticular waxes, but it has no impact on flower cuticle ultrastructure and cutin content. Expression analysis confirmed high transcription levels of SlCER6 in the anther and the petal, preferentially in sites subject to epidermal fusion. Hence, wax deficiency was proposed to be the primary reason for the flower fusion phenomenon in tomato. The SlCER6 substrate specificity was revisited. It might be involved in elongation of not only linear but also branched very-long-chain fatty acids, leading to production of the corresponding alkanes. SlCER6 implements a function in the sexual reproduction of tomato that is different from the one in Arabidopsis: SlCER6 is essential for the regulation of timely tapetum degradation and, consequently, microgametogenesis.

ETOILE regulates developmental patterning in the filamentous brown alga Ectocarpus siliculosus.

Brown algae are multicellular marine organisms evolutionarily distant from both metazoans and land plants. The molecular or cellular mechanisms that govern the developmental patterning in brown algae are poorly characterized. Here, we report the first morphogenetic mutant, étoile (etl), produced in the brown algal model Ectocarpus siliculosus. Genetic, cellular, and morphometric analyses showed that a single recessive locus, ETL, regulates cell differentiation: etl cells display thickening of the extracellular matrix (ECM), and the elongated, apical, and actively dividing E cells are underrepresented. As a result of this defect, the overrepresentation of round, branch-initiating R cells in the etl mutant leads to the rapid induction of the branching process at the expense of the uniaxial growth in the primary filament. Computational modeling allowed the simulation of the etl mutant phenotype by including a modified response to the neighborhood information in the division rules used to specify wild-type development. Microarray experiments supported the hypothesis of a defect in cell-cell communication, as primarily Lin-Notch-domain transmembrane proteins, which share similarities with metazoan Notch proteins involved in binary cell differentiation were repressed in etl. Thus, our study highlights the role of the ECM and of novel transmembrane proteins in cell-cell communication during the establishment of the developmental pattern in this brown alga.

The Arabidopsis dynamin-related protein2 family is essential for gametophyte development.

Clathrin-mediated membrane trafficking is critical for multiple stages of plant growth and development. One key component of clathrin-mediated trafficking in animals is dynamin, a polymerizing GTPase that plays both regulatory and mechanical roles. Other eukaryotes use various dynamin-related proteins (DRP) in clathrin-mediated trafficking. Plants are unique in the apparent involvement of both a family of classical dynamins (DRP2) and a family of dynamin-related proteins (DRP1) in clathrin-mediated membrane trafficking. Our analysis of drp2 insertional mutants demonstrates that, similar to the DRP1 family, the DRP2 family is essential for Arabidopsis thaliana development. Gametophytes lacking both DRP2A and DRP2B were inviable, arresting prior to the first mitotic division in both male and female gametogenesis. Mutant pollen displayed a variety of defects, including branched or irregular cell plates, altered Golgi morphology and ectopic callose deposition. Ectopic callose deposition was also visible in the pollen-lethal drp1c-1 mutant and appears to be a specific feature of pollen-defective mutants with impaired membrane trafficking. However, drp2ab pollen arrested at earlier stages in development than drp1c-1 pollen and did not accumulate excess plasma membrane or display other gross defects in plasma membrane morphology. Therefore, the DRP2 family, but not DRP1C, is necessary for cell cycle progression during early gametophyte development. This suggests a possible role for DRP2-dependent clathrin-mediated trafficking in the transduction of developmental signals in the gametophyte.

Dehydration protection provided by a maternal cuticle improves offspring fitness in the moss Funaria hygrometrica.

BACKGROUND AND AIMS: In bryophytes the sporophyte offspring are in contact with, nourished from, and partially surrounded by the maternal gametophyte throughout their lifespan. During early development, the moss sporophyte is covered by the calyptra, a cap of maternal gametophyte tissue that has a multilayered cuticle. In this study the effects on sporophyte offspring fitness of removing the maternal calyptra cuticle, in combination with dehydration stress, is experimentally determined. METHODS: Using the moss Funaria hygrometrica, calyptra cuticle waxes were removed by chemical extraction and individuals were exposed to a short-term dehydration event. Sporophytes were returned to high humidity to complete development and then aspects of sporophyte survival, development, functional morphology, and reproductive output were measured. KEY RESULTS: It was found that removal of calyptra cuticle under low humidity results in significant negative impacts to moss sporophyte fitness, resulting in decreased survival, increased tissue damage, incomplete sporophyte development, more peristome malformations, and decreased reproductive output. CONCLUSIONS: This study represents the strongest evidence to date that the structure of the calyptra cuticle functions in dehydration protection of the immature moss sporophyte. The investment in a maternal calyptra with a multilayered cuticle increases offspring fitness and provides a functional explanation for calyptra retention across mosses. The moss calyptra may represent the earliest occurance of maternal protection via structural provisioning of a cuticle in green plants.

Microanatomy of the placenta of Lycopodium obscurum: novel design in an underground embryo.

BACKGROUND AND AIMS: Long-lived underground populations of mycoheterotrophic gametophytes and attached sporophytes at various developmental stages occur in lycophytes. Young underground sporophytes obtain carbon solely from the gametophyte and establish nutritional independence only after reaching the soil surface, which may take several years. This prolonged period of matrotrophy exceeds that of bryophytes. The foot is massive and provides the lifeline for sporophyte establishment, yet the fine structure of the placental region is unexplored in lycophytes with underground gametophytes. METHODS: Gametophytes with attached embryos/young sporophytes of Lycopodium obscurum were collected in nature, processed and examined by light and transmission electron microscopy. KEY RESULTS: Three ultrastructurally distinct regions were identified within a single foot of a sporophyte emerging from the soil. Young foot regions actively divide, and have direct contact with and show little differentiation from gametophyte cells. In unlobed foot areas, cells in both generations exhibit polarity in content and indicate unidirectional transport of carbon reserves into the foot toward the developing shoot and root. The foot has inconspicuous wall ingrowths. Highly lobed foot regions contain peripheral transfer cells with prominent wall ingrowths that absorb nutrients from degenerating gametophyte cells. CONCLUSIONS: Variability within a single placenta is consistent with an invasive and long-lived foot. The late appearance of wall ingrowths in transfer cells reflects this dynamic ever-growing embryo. Placental features in lycophytes are related to the unique reorientation of all embryonic regions during development. Small placentas with wall ingrowths in both generations characterize ephemeral embryos in green gametophytes, while short-lived and repositioning embryos of heterosporous taxa are devoid of transfer cells. Transfer cell evolution across embryophytes is riddled with homoplasy and reflects diverse patterns of embryology. Scrutiny of placental evolution must include consideration of nutritional status and life history strategies of the gametophyte and young sporophyte.

Arabidopsis SNARE protein SEC22 is essential for gametophyte development and maintenance of Golgi-stack integrity.

Membrane traffic contributes to plant growth and development. However, the functional significance of SNARE proteins involved in membrane fusion of the early secretory pathway has not been explored with respect to plant development. Here we analyze the Arabidopsis v-SNARE SEC22. Loss of SEC22 function impairs gametophyte development, as indicated by reciprocal crosses between wild-type plants and plants heterozygous for T-DNA insertions in the SEC22 gene. sec22 mutant pollen becomes abnormal during the bicellular stage, eventually giving rise to degenerated pollen grains. Most mutant embryo sacs fail to support embryogenesis and display unfused polar nuclei in their central cell. Immunolocalization by both light and electron microscopy revealed an association of mutant-complementing Myc-tagged SEC22 with the central and peripheral endoplasmic reticulum (ER). Ultrastructural analysis of developing sec22 mutant pollen demonstrated Golgi fragmentation and consumption. As a consequence, the plasma membrane-targeted syntaxin SYP124 was retained in the ER. Our results suggest that SEC22 plays an essential role in early secretory traffic between the ER and the Golgi.

Fertility in barley flowers depends on Jekyll functions in male and female sporophytes.

• Owing to its evolutional plasticity and adaptability, barley (Hordeum vulgare) is one of the most widespread crops in the world. Despite this evolutionary success, sexual reproduction of small grain cereals is poorly investigated, making discovery of novel genes and functions a challenging priority. Barley gene Jekyll appears to be a key player in grain development; however, its role in flowers has remained unknown. • Here, we studied RNAi lines of barley, where Jekyll expression was repressed to different extents. The impact of Jekyll on flower development was evaluated based on differential gene expression analysis applied to anthers and gynoecia of wildtype and transgenic plants, as well as using isotope labeling experiments, hormone analysis, immunogold- and TUNEL-assays and in situ hybridization. • Jekyll is expressed in nurse tissues mediating gametophyte-sporophyte interaction in anthers and gynoecia, where JEKYLL was found within the intracellular membranes. The repression of Jekyll impaired pollen maturation, anther dehiscence and induced a significant loss of fertility. The presence of JEKYLL on the pollen surface also hints at possible involvement in the fertilization process. • We conclude that the role of Jekyll in cereal sexual reproduction is clearly much broader than has been hitherto realized.

Ultrastructural and cytochemical studies on oogenesis of the fern Pteridium aquilinum.

Egg development in Pteridium aquilinum var. latiusculum was studied using ultrastructural and cytochemical methods to examine structural features influencing fertilization in leptosporangiate ferns. Ultrastructural observations indicate a separation cavity is first formed above the egg during oogenesis with a pore region persistently connecting the egg and the ventral canal cell. The egg envelope is formed by deposition of amorphous materials in the separation cavity on the outer surface of plasmalemma. The egg envelope was not formed across the pore region; instead, a fertilization pore was formed. During oogenesis, the egg nucleus produced extensive evaginations containing osmiophilic bodies. Cytochemical experiments revealed that the egg envelope displays strong periodic acid-Schiff reaction indicative of polysaccharides, with negligible Sudan black B staining for lipids, suggesting that the egg envelope is composed principally of polysaccharides, and not lipids. The present manuscript provides new insights into egg structure and development of Pteridium, including discovery and characterization of the fertilization pore and observations on the chemical nature of the egg envelope, thus contributing to the understanding of the cytological mechanism of the sexual reproduction of ferns.