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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. [ABSTRACT FROM AUTHOR]

Transfer cells are characterized by an amplified plasma membrane area supported on a wall labyrinth composed of a uniform wall layer (UWL) from which wall ingrowth (WI) papillae arise. Adaxial epidermal cells of developing Vicia faba cotyledons, when placed in culture, undergo a rapid (hours) trans -differentiation to a functional epidermal transfer cell (ETC) phenotype. The trans -differentiation event is controlled by a signalling cascade comprising auxin, ethylene, apoplasmic reactive oxygen species (apoROS), and cytosolic Ca2+. Apoplasmic hydrogen peroxide (apoH2O2) was confirmed as the apoROS regulating UWL and WI papillae formation. Informed by an ETC-specific transcriptome, a pharmacological approach identified a temporally changing cohort of H2O2 biosynthetic enzymes. The cohort contained a respiratory burst oxidase homologue, polyamine oxidase, copper amine oxidase, and a suite of class III peroxidases. Collectively these generated two consecutive bursts in apoH2O2 production. Spatial organization of biosynthetic/catabolic enzymes was deduced from responses to pharmacologically blocking their activities on the cellular and subcellular distribution of apoH2O2. The findings were consistent with catalase activity constraining the apoH2O2 signal to the outer periclinal wall of the ETCs. Strategic positioning of class III peroxidases in this outer domain shaped subcellular apoH2O2 signatures that differed during assembly of the UWL and WI papillae. [ABSTRACT FROM AUTHOR]

Following similar studies of cell wall constituents in the placenta of Phaeoceros and Marchantia, we conducted immunogold labeling TEM studies of Physcomitrium patens to determine the composition of cell wall polymers in transfer cells on both sides of the placenta. Sixteen monoclonal antibodies were used to localize cell wall epitopes in the basal walls and wall ingrowths in this moss. In general, placental transfer cell walls of P. patens contained fewer pectins and far fewer arabinogalactan proteins AGPs than those of the hornwort and liverwort. P. patens also lacked the differential labeling that is pronounced between generations in the other bryophytes. In contrast, transfer cell walls on either side of the placenta of P. patens were relatively similar in composition, with slight variation in homogalacturonan HG pectins. Compositional similarities between wall ingrowths and primary cell walls in P. patens suggest that wall ingrowths may simply be extensions of the primary cell wall. Considerable variability in occurrence, abundance, and types of polymers among the three bryophytes and between the two generations suggested that similarity in function and morphology of cell walls does not require a common cell wall composition. We propose that the specific developmental and life history traits of these plants may provide even more important clues in understanding the basis for these differences. This study significantly builds on our knowledge of cell wall composition in bryophytes in general and in transfer cells across plants.

Transfer cells (TCs) facilitate high rates of nutrient transport into, and within, the plant body. Their transport function is conferred by polarized wall ingrowth papillae, deposited upon a specialized uniform wall layer, that form a scaffold supporting an amplified area of plasma membrane enriched in nutrient transporters. We explored the question of whether lipid-enriched domains of the TC plasma membrane could serve as organizational platforms for proteins regulating the construction of the intricate TC wall labyrinth using developing Vicia faba cotyledons. When these cotyledons are placed in culture, their adaxial epidermal cells trans -differentiate to a TC phenotype regulated by auxin, ethylene, extracellular hydrogen peroxide (apoH2O2), and cytosolic Ca2+ ([Ca2+]cyt) arranged in series. Staining cultured cotyledons with the sterol-specific dye, Filipin III, detected a polarized sterol-enriched domain in the plasma membrane of their trans- differentiating epidermal transfer cells (ETCs). Ethylene activated sterol biosynthesis while extracellular apoH2O2 directed sterol-enriched vesicles to fuse with the outer periclinal region of the ETC plasma membrane. The sterol-enriched domain was essential for generating the [Ca2+]cyt signal and orchestrating construction of both the uniform wall layer and wall ingrowth papillae. A model is presented outlining how the sterol-enriched plasma membrane domain forms and functions to regulate wall labyrinth assembly. [ABSTRACT FROM AUTHOR]

Transfer cells are characterized by wall labyrinths with either a flange or reticulate architecture. A literature survey established that reticulate wall ingrowth papillae ubiquitously arise from a modified component of their wall labyrinth, termed the uniform wall layer; a structure absent from flange transfer cells. This finding sparked an investigation of the deposition characteristics and role of the uniform wall layer using a Vicia faba cotyledon culture system. On transfer of cotyledons to culture, their adaxial epidermal cells spontaneously trans-differentiate to a reticulate architecture comparable to their abaxial epidermal transfer cell counterparts formed in planta. Uniform wall layer construction commenced once adaxial epidermal cell expansion had ceased to overlay the original outer periclinal wall on its inner surface. In contrast to the dense ring-like lattice of cellulose microfibrils in the original primary wall, the uniform wall layer was characterized by a sparsely dispersed array of linear cellulose microfibrils. A re-modeled cortical microtubule array exerted no influence on uniform wall layer formation or on its cellulose microfibril organization. Surprisingly, formation of the uniform wall layer was not dependent upon depositing a cellulose scaffold. In contrast, uniform wall cellulose microfibrils were essential precursors for constructing wall ingrowth papillae. On converging to form wall ingrowth papillae, the cellulose microfibril diameters increased 3-fold. This event correlated with up-regulated differential, and transfer-cell specific, expression of VfCesA3B while transcript levels of other cellulose biosynthetic-related genes linked with primary wall construction were substantially down-regulated.

Transfer cells (TCs) are anatomically-specialized cells formed at apoplasmic-symplasmic bottlenecks in nutrient transport pathways in plants. TCs form invaginated wall ingrowths which provide a scaffold to amplify plasma membrane surface area and thus increase the density of nutrient transporters required to achieve enhanced nutrient flow across these bottlenecks. Despite their importance to nutrient transport in plants, little is known of the transcriptional regulation of wall ingrowth formation. Here, we used RNA-Seq to identify transcription factors putatively involved in regulating epidermal TC development in cotyledons of Vicia faba. Comparing cotyledons cultured for 0, 3, 9 and 24 h to induce trans-differentiation of epidermal TCs identified 43 transcription factors that showed either epidermal-specific or epidermal–enhanced expression, and 10 that showed epidermal-specific down regulation. Members of the WRKY and ethylene-responsive families were prominent in the cohort of transcription factors showing epidermal-specific or epidermal–enhanced expression, consistent with the initiation of TC development often representing a response to stress. Members of the MYB family were also prominent in these categories, including orthologs of MYB genes involved in localized secondary wall deposition in Arabidopsis thaliana. Among the group of transcription factors showing down regulation were various homeobox genes and members of the MADs-box and zinc-finger families of poorly defined functions. Collectively, this study identified several transcription factors showing expression characteristics and orthologous functions that indicate likely participation in transcriptional regulation of epidermal TC development in V. faba cotyledons.

Trabalhos taxonômicos e filogenéticos têm utilizado informações anatômicas e para contribuir com estes estudos examinaram-se cortes paradérmicos e transversais da porção mediana foliar de Campomanesia adamantium (Camb.) O. Berg, Myrcia cordiifolia DC., M. decrescens O. Berg e M. torta D.C. Os caracteres anatômicos foram comparados com os de outras espécies descritas na literatura. São características comuns às quatro espécies examinadas: epiderme uniestratificada, tricomas unicelulares, folhas hipoestomáticas, estômatos paracíticos, cavidades secretoras em ambas as faces, mesofilo dorsiventral e nervura mediana com feixes bicolaterais. Destacam-se os seguintes caracteres úteis para a taxonomia da família: formato das células comuns da epiderme, tricomas dibraquiados ou não, camadas celulares incolores subepidérmicas e formato da nervura mediana. O formato e número das células teto das glândulas foliares têm utilidade taxonômica. O exame de 144 exsicatas evidenciou que a glabrescência é um fenômeno comum. A queda dos tricomas pode ser devido à sua base estreita e à ausência de célula pedal nos mesmos. Analisaram-se os caracteres à luz de estudos filogenéticos recentes e do ponto de vista ecológico, destacando a hipótese relacionada às células epidérmicas com possível função de célula de transferência. As características anatômicas forneceram dados para análises comparativas mais amplas entre os táxons de Myrtaceae e possibilitaram a construção de um cladograma, onde espécies de Leptospermum e Eucalyptus ocuparam posição basal, em consonância com filogenias relatadas na literatura.Taxonomical and phylogenetical studies have used anatomical information. In order to contribute to these studies, paradermal and cross sections of the median leaf blade portion of Campomanesia adamantium (Camb.) O. Berg, Myrcia cordiifolia DC., M. decrescens O. Berg and M. torta D.C. were examined. The anatomical characters were compared to the other species described in the literature. Some characteristics were irrespective of the four examined species: unistratified epidermis, unicellular trichomes, hypostomatic leaves, paracytic stomata, oil glands on both sides, dorsiventral mesophyll and bicollateral bundles on the midrib. The following characters appear to be taxonomically useful in the family: epidermal common cell format, presence of dibrachiate trichomes, presence of colorless subepidermal cell layers and midrib shape. The morphology and number of overlying cells associated with the glands have taxonomic utility. The 144 exsicates examination showed that the glabrescence is a widespread phenomenon in the Myrtaceae. The trichome fall can be due to its narrow base and to the foot cell absence. The characters were analyzed in the light of recent phylogenetic studies, highlighting the hypothesis related to the presence of epidermal cells with possible transfer cell function. The anatomical features provided data for wider comparative analysis amongst Myrtaceae taxa and ensured the construction of a cladogram, where Leptospermum and Eucalyptus species occupied the basal position, in line with reported phylogenies.

Vicia faba (L.) is an important cool-season grain legume species used widely in agriculture but also in plant physiology research, particularly as an experimental model to study transfer cell (TC) development. Adaxial epidermal cells of isolated cotyledons can be induced to form functional TCs, thus providing a valuable experimental system to investigate genetic regulation of TC development. The genome of V. faba is exceedingly large (ca. 13 Gb), however, and limited genomic information is available for this species. To provide a resource for transcript profiling of epidermal TC development, we have undertaken de novo assembly of a cotyledon-enriched transcriptome map for V. faba. Illumina paired-end sequencing of total RNA pooled from different tissues and different stages, including isolated cotyledons induced to form TCs, generated 69.5M reads, of which 65.8M were used for assembly following trimming and quality control. Assembly using a De-Bruijn graph-based approach within CLC Genomics Workbench v6.1 generated 21,297 contigs, of which 80.6% were successfully annotated against GO terms. The assembly was validated against known V. faba cDNAs held in GenBank, including transcripts previously identified as being specifically expressed in epidermal cells across TC trans-differentiation. This cotyledon-enriched transcriptome map therefore provides a valuable tool for future transcript profiling of epidermal TC development, and also enriches the genetic resources available for this important legume crop species.

Transfer cells (TCs) are anatomically-specialized cells formed at apoplasmic-symplasmic bottlenecks in nutrient transport pathways in plants. TCs form invaginated wall ingrowths which provide a scaffold to amplify plasma membrane surface area and thus increase the density of nutrient transporters required to achieve enhanced nutrient flow across these bottlenecks. Despite their importance to nutrient transport in plants, little is known of the transcriptional regulation of wall ingrowth formation. Here, we used RNA-Seq to identify transcription factors putatively involved in regulating epidermal TC development in cotyledons of Vicia faba. Comparing cotyledons cultured for 0, 3, 9, and 24 h to induce trans-differentiation of epidermal TCs identified 43 transcription factors that showed either epidermal-specific or epidermal-enhanced expression, and 10 that showed epidermal-specific down regulation. Members of the WRKY and ethylene-responsive families were prominent in the cohort of transcription factors showing epidermal-specific or epidermal-enhanced expression, consistent with the initiation of TC development often representing a response to stress. Members of the MYB family were also prominent in these categories, including orthologs of MYB genes involved in localized secondary wall deposition in Arabidopsis thaliana. Among the group of transcription factors showing down regulation were various homeobox genes and members of the MADs-box and zinc-finger families of poorly defined functions. Collectively, this study identified several transcription factors showing expression characteristics and orthologous functions that indicate likely participation in transcriptional regulation of epidermal TC development in V. faba cotyledons. [ABSTRACT FROM AUTHOR]

Ultrastructural studies on the female gametophyte are restricted to species at relatively derived positions in the angiosperm phylogenetic tree. Therefore, this topic remains mostly unknown for the early-divergent lineages, in which a four-celled megagametophyte is common. Here, ultrastructure of the megagametophyte and micropylar nucellar epidermis was investigated in Cabomba caroliniana A. Gray (Cabombaceae), Nymphaea gardneriana Planch. and Victoria cruziana Orb. (Nymphaeaceae). The micropylar nucellar epidermis of the studied species differentiates into an epistase. These cells have metabolically active cytoplasm and thickened inner tangential walls. Epistase ultrastructure is compatible with a transfer cell specialization. This tissue may play an adaptive role in the secretion of chemotropic substances to direct the pollen tube growth toward the female gametophyte. The cytological characteristics of the female germ unit in members of Cabombaceae and Nymphaeaceae are generally similar to other angiosperms that develop a typical seven-celled, eight-nucleate female gametophyte; however, they differ in some specific points. In V . cruziana and N . gardneriana , the micropylar end of the synergids develops a rudimentary filiform apparatus with slight inward projections. By contrast, the synergids lack a filiform apparatus in C. caroliniana. Unlike most studied angiosperms, the filiform apparatus in the clade Cabombaceae-Nymphaeaceae is underdeveloped or absent, therefore character state transformations have occurred within basal angiosperms. The potential evolutionary shifts of this reproductive feature are highlighted. [ABSTRACT FROM AUTHOR]

Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca2+ levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane-microtubule inter-relationship is discussed. [ABSTRACT FROM AUTHOR]

The enhanced transport capability of transfer cells (TCs) arises from their ingrowth wall architecture comprised of a uniform wall on which wall ingrowths are deposited. The wall ingrowth papillae provide scaffolds to amplify plasma membranes that are enriched in nutrient transporters. Using Vicia faba cotyledons, whose adaxial epidermal cells spontaneously and rapidly (hours) undergo a synchronous TC trans-differentiation upon transfer to culture, has led to the discovery of a cascade of inductive signals orchestrating deposition of ingrowth wall papillae. Auxin-induced ethylene biosynthesis initiates the cascade. This in turn drives a burst in extracellular H2O2 production that triggers uniform wall deposition. Thereafter, a persistent and elevated cytosolic Ca2+ concentration, resulting from Ca2+ influx through plasma membrane Ca2+-permeable channels, generates a Ca2+ signal that directs formation of wall ingrowth papillae to specific loci. We now report how these Ca2+-permeable channels are regulated using the proportionate responses in cytosolic Ca2+ concentration as a proxy measure of their transport activity. Culturing cotyledons on various combinations of pharmacological agents allowed the regulatory influence of each upstream signal on Ca2+ channel activity to be evaluated. The findings demonstrated that Ca2+-permeable channel activity was insensitive to auxin, but up-regulated by ethylene through two independent routes. In one route ethylene acts directly on Ca2+-permeable channel activity at the transcriptional and post-translational levels, through an ethylene receptor- dependent pathway. The other route is mediated by an ethylene-induced production of extracellular H2O2 which then acts translationally and post-translationally to up-regulate Ca2+-permeable channel activity. A model describing the differential regulation of Ca2+-permeable channel activity is presented. [ABSTRACT FROM AUTHOR]

Background: Transfer cells are characterized by intricate ingrowth walls, comprising an uniform wall upon which wall ingrowths are deposited. The ingrowth wall forms a scaffold to support an amplified plasma membrane surface area enriched in membrane transporters that collectively confers transfer cells with an enhanced capacity for membrane transport at bottlenecks for apo-/symplasmic exchange of nutrients. However, the underlying molecular mechanisms regulating polarized construction of the ingrowth wall and membrane transporter profile are poorly understood. Results: An RNAseq study of an inducible epidermal transfer cell system in cultured Vicia faba cotyledons identified transfer cell specific transcriptomes associated with uniform wall and wall ingrowth deposition. All functional groups of genes examined were expressed before and following transition to a transfer cell fate. What changed were the isoform profiles of expressed genes within functional groups. Genes encoding ethylene and Ca2+ signal generation and transduction pathways were enriched during uniform wall construction. Auxin-and reactive oxygen species-related genes dominated during wall ingrowth formation and ABA genes were evenly expressed across ingrowth wall construction. Expression of genes encoding kinesins, formins and villins was consistent with reorganization of cytoskeletal components. Uniform wall and wall ingrowth specific expression of exocyst complex components and SNAREs suggested specific patterns of exocytosis while dynamin mediated endocytotic activity was consistent with establishing wall ingrowth loci. Key regulatory genes of biosynthetic pathways for sphingolipids and sterols were expressed across ingrowth wall construction. Transfer cell specific expression of cellulose synthases was absent. Rather xyloglucan, xylan and pectin biosynthetic genes were selectively expressed during uniform wall construction. More striking was expression of genes encoding enzymes for re-modelling/degradation of cellulose, xyloglucans, pectins and callose. Extensins dominated the cohort of expressed wall structural proteins and particularly so across wall ingrowth development. Ion transporters were selectively expressed throughout ingrowth wall development along with organic nitrogen transporters and a large group of ABC transporters. Sugar transporters were less represented. Conclusions: Pathways regulating signalling and intracellular organization were fine tuned whilst cell wall construction and membrane transporter profiles were altered substantially upon transiting to a transfer cell fate. Each phase of ingrowth wall construction was linked with unique cohorts of expressed genes. [ABSTRACT FROM AUTHOR]

Vicia faba (L.) is an important cool-season grain legume species used widely in agriculture but also in plant physiology research, particularly as an experimental model to study transfer cell (TC) development. TCs are specialized nutrient transport cells in plants, characterized by invaginated wall ingrowths with amplified plasma membrane surface area enriched with transporter proteins that facilitate nutrient transfer. Many TCs are formed by trans-differentiation from differentiated cells at apoplasmic/symplasmic boundaries in nutrient transport. Adaxial epidermal cells of isolated cotyledons can be induced to form functional TCs, thus providing a valuable experimental system to investigate genetic regulation of TC trans-differentiation. The genome of V. faba is exceedingly large (ca. 13 Gb), however, and limited genomic information is available for this species. To provide a resource for future transcript profiling of epidermal TC differentiation, we have undertaken de novo assembly of a genome-wide transcriptome map for V. faba. Illumina paired-end sequencing of total RNA pooled from different tissues and different stages, including isolated cotyledons induced to form epidermal TCs, generated 69.5Mreads, of which 65.8M were used for assembly following trimming and quality control. Assembly using a De-Bruijn graph-based approach generated 21,297 contigs, of which 80.6% were successfully annotated against GO terms. The assembly was validated against known V. faba cDNAs held in GenBank, including transcripts previously identified as being specifically expressed in epidermal cells across TC trans-differentiation. This genome-wide transcriptome map therefore provides a valuable tool for future transcript profiling of epidermal TC trans-differentiation, and also enriches the genetic resources available for this important legume crop species. [ABSTRACT FROM AUTHOR]

Basal endosperm transfer layer (BETL) cells are responsible for transferring apoplastic solutes from the maternal pedicel into the endosperm, supplying the grain with compounds required for embryo development and storage reserve accumulation. Here, we analyze the maize (Zea mays L.) empty pericarp6 (emp6) mutant, which causes early arrest in grain development. The Emp6+gene function is required independently in both the embryo and endosperm. The emp6 mutant causes a notable effect on the differentiation of BETL cells; the extensive cell wall ingrowths that distinguish BETL cells are diminished and BETL marker gene expression is compromised in mutant kernels. Transposon tagging identified the emp6 locus as encoding a putative plant organelle RNA recognition (PORR) protein, 1 of 15 PORR family members in maize. The emp6 transcript is widely detected in plant tissues with highest levels in embryos and developing kernels. EMP6+green fluorescent protein (GFP) fusion proteins transiently expressed in Nicotiana benthamiana leaves were targeted specifically to mitochondria. These results suggest that BETL cell differentiation might be particularly energy intensive, or alternatively, that mitochondria might confer a developmental function. [ABSTRACT FROM AUTHOR]

A persistent and polarized cytosolic Ca2+ signal, formed into plumes by co-operative activities of plasma membrane Ca2+ channels and Ca2+-ATPase clusters, directs papillate wall ingrowth deposition in trans-differentiating transfer cells.Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall upon which wall ingrowth papillae develop at right angles into the cytoplasm. The hypothesis that positional information directing construction of wall ingrowth papillae is mediated by Ca2+ signals generated by spatiotemporal alterations in cytosolic Ca2+ ([Ca2+]cyt) of cells trans-differentiating to a transfer cell morphology was tested. This hypothesis was examined using Vicia faba cotyledons. On transferring cotyledons to culture, their adaxial epidermal cells synchronously trans-differentiate to epidermal transfer cells. A polarized and persistent Ca2+ signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca2+ signal intensity, by withdrawing extracellular Ca2+ or blocking Ca2+ channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca2+ signal polarity and persistence depended upon a rapid turnover (minutes) of cytosolic Ca2+ by co-operative functioning of plasma membrane Ca2+-permeable channels and Ca2+-ATPases. Viewed paradermally, and proximal to the cytosol–plasma membrane interface, the Ca2+ signal was organized into discrete patches that aligned spatially with clusters of Ca2+-permeable channels. Mathematical modelling demonstrated that these patches of cytosolic Ca2+ were consistent with inward-directed plumes of elevated [Ca2+]cyt. Plume formation depended upon an alternating distribution of Ca2+-permeable channels and Ca2+-ATPase clusters. On further inward diffusion, the Ca2+ plumes coalesced into a uniform Ca2+ signal. Blocking or dispersing the Ca2+ plumes inhibited deposition of wall ingrowth papillae, while uniform wall formation remained unaltered. A working model envisages that cytosolic Ca2+ plumes define the loci at which wall ingrowth papillae are deposited. [ABSTRACT FROM PUBLISHER]

Transfer cells (TCs) are ubiquitous throughout the plant kingdom. Their unique ingrowth wall labyrinths, supporting a plasma membrane enriched in transporter proteins, provides these cells with an enhanced membrane transport capacity for resources. In certain plant species, TCs have been shown to function to facilitate phloem loading and/or unloading at cellular sites of intense resource exchange between symplasmic/apoplasmic compartments. Within the phloem, the key cellular locations of TCs are leaf minor veins of collection phloem and stem nodes of transport phloem. In these locations, companion and phloem parenchyma cells trans-differentiate to a TC morphology consistent with facilitating loading and re-distribution of resources respectively. At a species level, occurrence of TCs is significantly higher in transport than in collection phloem. TCs are absent from release phloem but occur within post-sieve element unloading pathways and particularly at interfaces between generations of developing Angiosperm seeds. Experimental accessibility of seed TCs has provided opportunities to investigate their inductive signaling, regulation of ingrowth wall formation and membrane transport function. This review uses this information base to explore current knowledge of phloem transport function and inductive signaling for phloem-associated TCs. The functional role of collection phloem and seed TCs is supported by definitive evidence, but no such information is available for stem node TCs that present an almost intractable experimental challenge. There is an emerging understanding of inductive signals and signaling pathways responsible for initiating trans-differentiation to a TC morphology in developing seeds. However, scant information is available to comment on a potential role for inductive signals that induce seed TCs, in regulating induction of phloem-associated TCs. Biotic phloem invaders have been used as a model to speculate on involvement of these signals.

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. [ABSTRACT FROM AUTHOR]

Transfer cells (TCs) are ubiquitous throughout the plant kingdom. Their unique ingrowth wall labyrinths, supporting a plasma membrane enriched in transporter proteins, provides these cells with an enhanced membrane transport capacity for resources. In certain plant species, TCs have been shown to function to facilitate phloem loading and/or unloading at cellular sites of intense resource exchange between symplasmic/apoplasmic compartments. Within the phloem, the key cellular locations of TCs are leaf minor veins of collection phloem and stem nodes of transport phloem. In these locations, companion and phloem parenchyma cells trans-differentiate to a TC morphology consistent with facilitating loading and re-distribution of resources, respectively. At a species level, occurrence of TCs is significantly higher in transport than in collection phloem. TCs are absent from release phloem, but occur within post-sieve element unloading pathways and particularly at interfaces between generations of developing Angiosperm seeds. Experimental accessibility of seed TCs has provided opportunities to investigate their inductive signaling, regulation of ingrowth wall formation and membrane transport function. This review uses this information base to explore current knowledge of phloem transport function and inductive signaling for phloem-associated TCs. The functional role of collection phloem and seed TCs is supported by definitive evidence, but no such information is available for stem node TCs that present an almost intractable experimental challenge. There is an emerging understanding of inductive signals and signaling pathways responsible for initiating trans-differentiation to a TC morphology in developing seeds. However, scant information is available to comment on a potential role for inductive signals (auxin, ethylene and reactive oxygen species) that induce seed TCs, in regulating induction of phloem-associated TCs. Biotic phloem invaders have been used as a model to speculate on involvement of these signals. [ABSTRACT FROM AUTHOR]

Various cell types can trans-differentiate to a transfer cell (TC) morphology characterized by deposition of polarized ingrowth walls comprised of a uniform layer on which wall ingrowths (WIs) develop. WIs form scaffolds supporting amplified plasma membrane areas enriched in transporters conferring a cellular capacity for high rates of nutrient exchange across apo- and symplasmic interfaces. The hypothesis that reactive oxygen species (ROS) are a component of the regulatory pathway inducing ingrowth wall formation was tested using Vicia faba cotyledons. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, on being placed into culture, their adaxial epidermal cells rapidly (hours) form ingrowth walls on their outer periclinal walls. These are readily visualized by electron microscopy, and epidermal peels of their trans-differentiating cells allow measures of cell-specific gene expression. Ingrowth wall formation responded inversely to pharmacological manipulation of ROS levels, indicating that a flavin-containing enzyme (NADPH oxidase) and superoxide dismutase cooperatively generate a regulatory H2O2 signature. Extracellular H2O2 fluxes peaked prior to the appearance of WIs and were followed by a slower rise in H2O2 flux that occurred concomitantly, and co-localized, with ingrowth wall formation. De-localizing the H2O2 signature caused a corresponding de-localization of cell wall deposition. Temporal and epidermal cell-specific expression profiles of VfrbohA and VfrbohC coincided with those of extracellular H2O2 production and were regulated by cross-talk with ethylene. It is concluded that H2O2 functions, downstream of ethylene, to activate cell wall biosynthesis and direct polarized deposition of a uniform wall on which WIs form. [ABSTRACT FROM PUBLISHER]

The suspensor plays an active role during the early embryo development of flowering plants. In orchids, the suspensor cells are highly vacuolated without structural specializations, and the possible mechanism(s) that enable the suspensor to serve as the nutrient uptake site is virtually unknown. Here, we used the fluorescent tracer CFDA to characterize the pathway for symplastic transport in the suspensor cells of developing embryos and to provide direct visual evidence that the orchid suspensor has unique physiological properties. The embryo proper uptakes the fluorescent dye through the suspensor. CF could first be detected throughout the suspensor cell and then subsequently in the embryo proper. A plasmolysis experiment clearly indicates that suspensor cells have a more negative osmotic potential than the adjoining testa cells. It is proposed that the preferential entry of CFDA into the suspensor cell of the Nun orchid is aided by the more negative osmotic potential of the suspensor than neighboring cells, providing a driving force for the uptake of water from the apoplast into the symplast. [ABSTRACT FROM AUTHOR]

Transfer cells (TCs) are specialized cells exhibiting invaginated wall ingrowths (WIs), thereby amplifying their plasma membrane surface area (PMSA) and hence the capacity to transport nutrients. However, it remains unknown as to whether TCs play a role in biomass yield increase during evolution or domestication. Here, we examine this issue from a comparative evolutionary perspective. The cultivated tetraploid AD genome species of cotton and its A and D genome diploid progenitors displayed high, medium, and low seed and fiber biomass yield, respectively. In all three species, cells of the innermost layer of the seed coat juxtaposed to the filial tissues trans-differentiated to a TC morphology. Electron microscopic analyses revealed that these TCs are characterized by sequential formation of flange and reticulate WIs during the phase of rapid increase in seed biomass. Significantly, TCs from the tetraploid species developed substantially more flange and reticulate WIs and exhibited a higher degree of reticulate WI formation than their progenitors. Consequently, the estimated PMSA of TCs of the tetraploid species was about 4 and 70 times higher than that of TCs of the A and D genome progenitors, respectively, which correlates positively with seed and fiber biomass yield. Further, TCs with extensive WIs in the tetraploid species had much stronger expression of sucrose synthase, a key enzyme involved in TC WI formation and function, than those from the A and D progenitors. The analyses provide a set of novel evidence that the development of TC WIs may play an important role in the increase of seed and fiber biomass yield through polyploidization during evolution. [ABSTRACT FROM PUBLISHER]

This is the first review of the developmental morphology of pteridophyte embryos since molecular phylogenies revolutionized concepts of tracheophyte evolution. In the light of these novel relationships, embryo characters are evaluated across pteridophytes to hypothesize homology and infer character transformations. Salient features of traditional categories used to classify pteridophyte embryos, for example endoscopy and exoscopy, are analyzed and related to gametophyte habit. Suspensor formation evolved in several lineages and is reinterpreted as representing a distinct developmental stage of the foot which is the only embryonic organ that is homologous across all land plants. Endoscopy in lycophytes is distinct from that of monilophytes, because lycophyte embryos undergo reorientation, lack interdigitating placental cells, and contain an interplacental space which may be related to their lack of a basal pad cell during archegonial development. Pteridophyte embryology may provide clues to tracheophytes evolution and novel developmental mechanisms. [ABSTRACT FROM AUTHOR]

This comprehensive study of early embryology in Ceratopteris richardii combines light microscopy with the first ultrastructural evaluation of any pteridophyte embryo. Emphasis is placed on ontogeny of the foot and placental transfer cells. The embryology of C. richardii shares many similarities with that of other polypodiacious ferns while exhibiting distinctive division patterns. Formative embryonic stages have been reconstructed into three-dimensional models for ease of interpretation. The zygote divides perpendicular to the gametophyte plane and anterioposterior axis. This division establishes a prone embryological habit that maximizes rapid independent establishment of a leaf-root axis in a cordate gametophyte. After the formation of a globular eight-celled stage, initials of the first leaf, and root and shoot apical meristems are defined early by discrete formative divisions. Concomitantly, the foot expands and differentiates to transport nutrients from the gametophyte for the developing embryonic organs. Transfer cell wall ingrowth deposition begins in the gametophyte placental cells before the adjacent sporophyte cells just after the eight-celled stage. These observations provide an anatomical framework for future comparative developmental genetic studies of embryogenesis in free-sporing plants. [ABSTRACT FROM AUTHOR]

Data from this study show that the chalazal cyst is a consistent feature of the Brassicaceae that exhibits variation of potential taxonomic value. The U-shaped seeds of mustards comprise three development chambers: micropylar (MC), central (CC), and chalazal (ChC). Early in seed development the syncytial endosperm in the ChC becomes differentiated into a cyst that remains distinct from storage endosperm. In early syncytial stages, the cyst is stratified into three zones: (1) an apical zone containing nuclei, plastids and mitochondria, (2) a mid-zone rich in endomembranes but depleted of large organelles, and (3) a basal portion that is bordered by a labyrinthine wall. The basal zone has projections that range from foot-like extensions to thread-like haustoria that penetrate the underlying maternal chalazal proliferative tissues (CPT). No cell-to-cell connections occur between endosperm and maternal tissue, but the extensive labyrinthine walls closely invest cell lysate resulting from degradation of the CPT. Structural data from light, confocal and transmission electron microscopy suggest that the cyst is active in uptake and transport of metabolites into the developing seed. Four types of cyst reflecting position and size of the ChC are recognized. In general, they correlate with major tribes of Brassicaceae. Data support recent molecular studies indicating that classification of the Lepidieae is artificial and suggest that Thlaspi is a candidate for realignment. The most divergent morphology in a tribe is seen in the two species of the Hesperidae. Erysimum has a more typical chalazal chamber and cyst morphology while the tubular chalazal chamber and filiform cyst of Chorispora is of the most extreme type encountered in the survey. This suggests that the tribe is paraphyletic and should be reinvestigated to clarify relationships. © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 144, 375−394. [ABSTRACT FROM AUTHOR]

Eriophorum spp. are abundant perennial graminoids in the Arctic tundra and boreal peatlands. Because ecological studies indicated that some plants are unusually productive on infertile and cold sites, the anatomy of the overwintering corms of Eriophorum vaginatum (L.) and Eriophorum scheuchzeri (Hoppe) were examined to determine their involvement in nutrient uptake and storage. Components of the long‐distance transport pathways were identified within the plants by using histochemical techniques and transport of apoplastic and symplastic dyes. E. scheuchzeri produced a rhizome that consisted mainly of storage parenchyma cells within which collateral vascular bundles were centrally located and arranged in a circle. By contrast, E. vaginatum developed a ring of horizontally arranged xylem and phloem, in addition to axial amphivasal vascular bundles leading to the leaves, all of which were bordered by transfer cells. As shown by the transport of fluorescein in the phloem and Safranine O in the xylem, each axial bundle and adventitious root contacted the horizontal ring of vascular tissues so that solutes from one vascular bundle were translocated into the vascular ring and circulated to another vascular bundle and/or to the roots. In addition, special groups of sclereids that functioned in both phloem and xylem transport were found at the base of the leaf traces and within junctions of senescing roots. These sclereids were named ‘vascular sclerenchyma’ and it was hypothesized that they provide a moving end for the vascular system because the corm dies progressively from the distal end as it grows upward from the apical meristem. It was concluded that this unusual vascular system of E. vaginatum is efficient in recycling nutrients internally, which may account for its competitive advantage in infertile and cold sites. [ABSTRACT FROM PUBLISHER]

ABSTRACT The immunocytochemical localization of the plasma membrane H+ -ATPase in epidermal cells of tomato roots was studied using a monoclonal antibody raised against purified maize P-type H+ -ATPase. Plants subjected to iron starvation exhibited increased proton extrusion that was confined to the root elongation zones. Immunogold labelling of the H+ -ATPase on the plasma membrane was considerably higher in rhizodermal cells within zones with intense proton extrusion than in non-acidifying areas of the roots. Transfer cells were formed in rhizodermal cells of Fe-deficient plants. Quantitative determination of immunolabelling revealed that the density of PM H+ -ATPase in transfer cells was about twice that of ordinary epidermal cells. In transfer cells, H+ -ATPase was most abundant on the plasma membrane lining the labyrinthine invaginations of the peripheral cell wall. While the number of immunologically detectable ATPase molecules in transfer cells was not spatially correlated with proton extrusion activity, the frequency of transfer cells was considerably higher in acidifying root areas relative to non-active segments. Split-root experiments indicated that both the steady-state level of plasma membrane H+ -ATPase and proton extrusion activity are systemically regulated, indicating inter-organ regulation of rhizosphere acidification. Exogenous application of the auxin analog 2,4-dichlorophenoxyacetic acid and the ethylene precursor 1-aminocyclopropane-1-carboxlic acid caused the formation of transfer cells at a frequency similar to that observed in Fe-deficient roots. However, the number of proton pumps was not affected by the hormone treatment, suggesting that both responses are regulated independently. It is concluded that transfer cells in the rhizodermis may be important but not crucial for rhizosphere acidification. [ABSTRACT FROM AUTHOR]

In roots of tomato (Lycopersicon esculentum Mill.), extranumerary root hairs and transfer cell-like wall ingrowth depositions in the rhizodermis were developed in response to P and Fe deficiency. Immunocytolocalization of the plasma membrane H+-ATPase in roots of P-deficient plants revealed no appreciable increase in H+-ATPase density relative to control plants. In transfer cells, immunogold labeling was considerably higher than in ordinary rhizodermal cells. H+-ATPase sites were asymmetrically distributed in cells with and without wall ingrowths under P-deficient conditions. A split-root study revealed that the frequency of transfer cells was higher in the low-P half of the root system, but the density of H+-ATPase molecules was enhanced only in the high-P half of the split roots, suggesting that formation of transfer cells was controlled directly by the external Pi concentration, whereas ATPase expression was regulated indirectly by the internal nutrient status of the plant. The role of hormones in the induction of transfer cells was investigated by treating plants with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or various ethylene antagonists. Transfer cells were induced by ACC to an extent similar to that observed after P or Fe starvation, but inhibitors of either ethylene synthesis or action did not decrease their frequency. These results suggest that ethylene was not required for the induction of transfer cells but changes in ethylene levels appeared to modulate the number of cells forming wall ingrowths. In roots of ethylene-insensitive Never-ripe tomato plants the frequency of transfer cells was rather increased than decreased under most growth conditions relative to the wild type, indicating that ethylene responsiveness played no critical role in the differentiation of transfer cells and that the transduction of signals ultimately leading to their formation was independent of the ethylene signaling cascade. [ABSTRACT FROM AUTHOR]

Transfer-cell-like wall ingrowth deposition is induced in adaxial epidermal cells of Vicia faba L. cotyledons grown in vitro in the presence of high hexose concentrations. We have further characterised this putative transfer cell induction system by examining initiation of secondary-wall ingrowth deposition and expression of sucrose transport-related genes. Wall ingrowth deposition, as wall thickening and scattered papillate ingrowths, was visualised on the outer periclinal walls of adaxial epidermal cells of cotyledons after 1 day in culture. Over the next 2 days, wall deposition increased significantly to form a distinct band of discrete and coalescing ingrowths. Thereafter, further wall deposition was arrested. Densities of Golgi, endoplasmic reticulum, and mitochondria increased concurrently with wall ingrowth deposition. Transcripts of a H/sucrose symporter (SUT) and a sucrose-binding protein (SBP) were detected by in situ hybridisation in differentiating transfer cells. Antibodies raised against an H-ATPase immunolocalised evenly around the perimeter of the adaxial epidermal cells in day 1 cotyledons. Thereafter, labelling became increasingly localised to the developing wall ingrowth regions. In contrast, SBP antibodies immunolocalised exclusively to wall ingrowth regions. However, SBP exhibited a transient pattern of expression, being detected only in 2-day-cultured cotyledons. A proton gradient, sufficient to facilitate sulphorhodamine G accumulation, was established by adaxial epidermal cells after 1 day in culture. [C]sucrose uptake by cotyledons became sensitive to an inhibitor of carrier-mediated transport of sucrose, para-chloromercuribenzene sulfonic acid after 2 days. The initial 37% inhibition of sucrose transport by this compound declined to 5% for cotyledons cultured for 3 days. Collectively, these results suggest that differentiation of the key functional characteristics of transfer cells can be induced in vitro, providing an exciting tool for further exploration of transfer cell development. [ABSTRACT FROM AUTHOR]

Following similar studies of cell wall constituents in the placenta of Phaeoceros and Marchantia, we conducted immunogold labeling TEM studies of Physcomitrium patens to determine the composition of cell wall polymers in transfer cells on both sides of the placenta. Sixteen monoclonal antibodies were used to localize cell wall epitopes in the basal walls and wall ingrowths in this moss. In general, placental transfer cell walls of P. patens contained fewer pectins and far fewer arabinogalactan proteins AGPs than those of the hornwort and liverwort. P. patens also lacked the differential labeling that is pronounced between generations in the other bryophytes. In contrast, transfer cell walls on either side of the placenta of P. patens were relatively similar in composition, with slight variation in homogalacturonan HG pectins. Compositional similarities between wall ingrowths and primary cell walls in P. patens suggest that wall ingrowths may simply be extensions of the primary cell wall. Considerable variability in occurrence, abundance, and types of polymers among the three bryophytes and between the two generations suggested that similarity in function and morphology of cell walls does not require a common cell wall composition. We propose that the specific developmental and life history traits of these plants may provide even more important clues in understanding the basis for these differences. This study significantly builds on our knowledge of cell wall composition in bryophytes in general and in transfer cells across plants. [ABSTRACT FROM AUTHOR]

In maize, a layer of basal endosperm cells adjacent to the pedicel is modified for a function in solute transfer. Three genes specifically expressed in this region, termed the basal endosperm transfer layer (BETL-2 to -4), were isolated by differential hybridization. BETL-2 to -4 are coordinately expressed in early and mid-term endosperm development, but are absent at later stages. BETL-2 to -4 coding sequences all predict small (<100 amino acids), secreted, cysteine-rich polypeptides which lack close relatives in current database accessions. BETL-3 and BETL-1 display some sequence similarities with each other and to plant defensins. BETL-2 to -4 promoter regions were isolated and compared, revealing the presence of a promoter-proximal microsatellite repeat as the most highly conserved sequence element in each sequence. Electrophoretic mobility shift assays (EMSA) showed that specific BETL-2 to -4 promoter fragments competed for binding to the same DNA-binding activity in nuclear extracts prepared from maize endosperm. Although BETL-2 to -4 are only expressed in basal endosperm cells, the DNA-binding activities detected were of two types: distal endosperm-specific, or present in both basal and distal endosperm extracts. On the basis of these findings, a model to account for the coordinate regulation of BETL genes in endosperm cells is proposed. [ABSTRACT FROM AUTHOR]

Ultrastructural and histochemical studies reveal that apogamous sporophytes develop spontaneously from gametophytic callus cells of Physcomitrium coorgense which have accumulated starch and developed wall ingrowths. Plasmodesmatal connections are retained between the apogamous outgrowths and the subtending callus. Although apogamous sporophytes lack a recognizable foot, the peripheral cells in the lower part of the seta develop wall ingrowths similar to those in the placental region in sporophytes of sexual origin. [ABSTRACT FROM AUTHOR]

In the developing wheat grain, photosynthate is transferred longitudinally along the crease phloem and then laterally into the endosperm cavity through the crease vascular parenchyma, pigment strand and nucellar projection. In order to clarify this cellular pathway of photosynthate unloading, and hence the controlling mechanism of grain filling, the potential for symplastic and apoplastic transfer was examined through structural and histochemical studies on these tissue types. It was found that cells in the crease region from the phloem to the nucellar projection are interconnected by numerous plasmodesmata and have dense cytoplasm with abundant mitochondria. Histochemical studies confirmed that, at the stage of grain development studied, an apoplastic barrier exists in the cell walls of the pigment strand. This barrier is composed of lignin, phenolics and suberin. The potential capacity for symplastic transfer, determined by measuring plasmodesmatal frequencies and computing potential sucrose fluxes through these plasmodesmata, indicated that there is sufficient plasmodesmatal cross-sectional area to support symplastic unloading of photosynthate at the rate required for normal grain growth. The potential capacity for membrane transport of sucrose to the apoplast was assessed by measuring plasma membrane surface areas of the various cell types and computing potential plasma membrane fluxes of sucrose. These fluxes indicated that the combined plasma membrane surface areas of the sieve element--companion cell (se--cc) complexes, vascular parenchyma and pigment strand are not sufficient to allow sucrose transfer to the apoplast at the observed rates. In contrast, the wall ingrowths of the transfer cells in the nucellar projection amplify the membrane surface area up to 22-fold, supporting the observed rates of sucrose transfer into the endosperm cavity. We conclude that photosynthate moves via the symplast from the se--cc complexes to the nucellar projection transfer cells, from where it is transferred across the plasma membrane into the endosperm cavity. The apoplastic barrier in the pigment strand is considered to restrict solute movement to the symplast and block apoplastic solute exchange between maternal and embryonic tissues. The implications of this cellular pathway in relation to the control of photosynthate transfer in the developing grain are discussed. [ABSTRACT FROM AUTHOR]