Subepidermal Cell Layers Research Articles

AtSUC3, a gene encoding a new Arabidopsis sucrose transporter, is expressed in cells adjacent to the vascular tissue and in a carpel cell layer

The cDNA corresponding to the open reading frame T17M13.3 from Arabidopsis chromosome II was isolated and the encoded protein was characterized as a member of a subgroup of higher plant sucrose transporters. The AtSUC3 (Arabidopsis thaliana sucrose transporter 3) open reading frame encodes a protein with 594 amino acid residues, being 81 and 82 residues longer than the previously described Arabidopsis sucrose carriers AtSUC1 and AtSUC2. About 50 of these additional amino acids are part of an extended cytoplasmic loop separating the N-terminal from the C-terminal half of the protein. For functional characterization the AtSUC3 cDNA was expressed in baker's yeast. Substrate specificities, energy dependence and K(m) values of the recombinant protein were determined. Removal of the enlarged cytoplasmic loop and expression of the truncated cDNA caused no detectable change in the kinetic properties of the protein, suggesting a transport-independent function for this cytoplasmic domain. Immunolocalization with an AtSUC3-specific antiserum identified the protein in a cell layer separating the phloem from the mesophyll and in a single, subepidermal cell layer of the carpels that is important for pod dehiscence. These localizations suggest a possible role of AtSUC3 in the funnelling of sucrose from the mesophyll towards the phloem, and possibly in pod shatter.

AtSUC3, a gene encoding a new Arabidopsis sucrose transporter, is expressed in cells adjacent to the vascular tissue and in a carpel cell layer

SummaryThe cDNA corresponding to the open reading frame T17M13.3 from Arabidopsis chromosome II was isolated and the encoded protein was characterized as a member of a subgroup of higher plant sucrose transporters. The AtSUC3 (Arabidopsis thaliana sucrose transporter 3) open reading frame encodes a protein with 594 amino acid residues, being 81 and 82 residues longer than the previously described Arabidopsis sucrose carriers AtSUC1 and AtSUC2. About 50 of these additional amino acids are part of an extended cytoplasmic loop separating the N‐terminal from the C‐terminal half of the protein. For functional characterization the AtSUC3 cDNA was expressed in baker's yeast. Substrate specificities, energy dependence and Km values of the recombinant protein were determined. Removal of the enlarged cytoplasmic loop and expression of the truncated cDNA caused no detectable change in the kinetic properties of the protein, suggesting a transport‐independent function for this cytoplasmic domain. Immunolocalization with an AtSUC3‐specific antiserum identified the protein in a cell layer separating the phloem from the mesophyll and in a single, subepidermal cell layer of the carpels that is important for pod dehiscence. These localizations suggest a possible role of AtSUC3 in the funnelling of sucrose from the mesophyll towards the phloem, and possibly in pod shatter.

A kinetic model for turgor loss in red cabbage cells during mild heat treatment

Summary Turgor changes in red cabbage leaves (Brassica oleracea L. covar. Capitata Alef. var. rubra DC.) due to mild heat treatments were measured in the epidermal and first subepidermal cell layers with the pressure probe technique. After an equilibration time in a sorbitol solution of equal water potential, cabbage discs were immersed in solutions heated to 53 °C and 65 °C for different time intervals. Turgor loss at both temperatures could be described by a first order degradation model. This could be due to the increased permeability of membranes at higher temperatures, resulting in passive efflux of solutes and an irreversible reduction of cell turgor well before membranes are completely destroyed. The residual turgor (at t = ∞) for the 53 °C treatment was significantly different from zero. Assuming an Arrhenius type model for the temperature dependency of the rate constant k, the activation energy (Ea = 41 kJ · mol-1) and reference rate constant (kref = 1.53 min-1 for a reference temperature Tref = 323 K) were calculated for the turgor loss model in the epidermal cell layer. In the second cell layer, a time shift of the commencement of turgor decrease could be observed at the lower processing temperature. Temperature profiles at different locations in the cabbage disc were calculated by use of a finite element model and related to turgor loss observations.

A comparative structural analysis of direct and indirect shoot regeneration of Papaver somniferum L. in vitro

Summary Cellular origin of shoot buds, cell morphogenesis and differentiation were studied during direct and indirect shoot regeneration of Papaver somniferum L. in vitro . Direct shoot organogenesis was induced in immature somatic embryos, where cell division and protomeristem formation started in sub-epidermal and epidermal cell layers of hypocotyl. Indirect shoot regeneration was initiated from callus culture using auxins and cytokinins, where compact globular meristemoids were produced. The common morphogenetic event of direct and indirect shoot regeneration was an establishment of non-random cell division and restricted cell expansion within the group of competent cells during protomeristem formation. However, in contrast to direct regeneration, where all activated cells became competent, in indirect regeneration, only peripheral cells of meristemoids acquired morphogenetic competence. The second difference occurred in shoot tunica formation: original hypocotyl epidermal cells participated in tunica formation during direct organogenesis, while this layer regenerated de novo in meristemoids. These results indicate that cell morphogenesis during shoot regeneration is independent of the developmental history of the competent cells.

In vitro regeneration of Echinacea purpurea L.: Direct somatic embryogenesis and indirect shoot organogenesis in petiole culture

An in vitro propagation system was developed for Echinacea purpurea L. (purple coneflower), a medicinal plant commonly used in the treatment of colds, flu and related ailments. Echinacea seeds were found to be contaminated with systemic fungi and therefore an optimized minimal concentration of Plant Preservation Mixture (PPM) was incorporated in the seed germination medium to recover sterile seedlings. Regeneration was induced on petiole explants from 2-month-old sterile seedlings cultured on medium supplemented with benzylaminopurine (BAP) or thidiazuron (TDZ) in combination with indoleacetic acid (IAA). Two distinct forms of regeneration were identified in cultured petiole explants with histological and morphological observations, viz. the direct formation of somatic embryos on the epidermis and the de novo development of shoots from callus tissues formed in subepidermal cell layers. the results of this study have established a micropropagation system for E. purpurea that will provide sterile plant material for further investigations into medicinally active biochemicals and may facilitate mass production of high-quality E. purpurea plants for the commercial market.

The First Stages of Pre-harvest ‘Peel Pitting’ Development in ‘Encore’ Mandarin. An Histological and Ultrastructural Study

‘Peel pitting’ in the flavedo (the coloured epicarp) of ‘Encore’ mandarin arises as chlorotic spots. These spots correspond to parenchymal flattening and collapse of a variable number of sub-epidermal cell layers, which increase in parallel with the epidermis, extending between apparently healthy zones. The first signs of cellular damage are associated with internal membrane disorganization of the plastids. Great vesiculation of the cytoplasm occurs, followed by degradation of cytoplasmic membranes and accumulation of abundant osmiophilic material, yielding amorphous and dense masses. The disorder started by September. Spotting was only observed on the outer side of the fruit and on portions of the rind most exposed to external factors. Radiation and high temperature of the rind over long periods may induce localized dehydration, plasmolysis and cell collapse.

Localization of cytokinins in somatic and zygotic embryos of Tilia cordata using immunocytochemistry

Immunofluorescent and immunogold labeling was used to study the localization of cytokinins in developing somatic and zygotic embryos of Tilia cordata Miller. Broad‐specificity polyclonal antibodies active against dihydrozeatin riboside (DHZR)/zeatin riboside (ZR)‐type and isopentenyladenosine (iPR)‐type cytokinins were used for immunolabeling. Immunofluorescent microscopy showed that these cytokinins were concentrated in highly cytoplasmic cells showing meristematic character. Cotyledon initials and primary meristems of heart‐stage somatic embryos, as well as heart‐stage zygotic embryos, were labeled. During elongation of embryos, cytokinin immunoreactive material was concentrated to areas having meristematic character. Root apex, shoot meristem, and cotyledon cells of somatic and zygotic cotyledonary embryos, as well as epidermal and subepidermal cell layers of the hypocotyl, showed the strongest immunoreaction. The nucleoli, especially, had a very strong signal. Results at the ultrastructural level with gold‐conjugated protein A supported these conclusions. Gold particles were distributed in the nuclei, especially in the nucleoli and throughout the ground cytoplasm. They were occasionally associated with plastids and mitochondria, but seldom with other organelles.

Developmentally regulated patterns of expression directed by poplar PAL promoters in transgenic tobacco and poplar.

Phenylalanine ammonia-lyase (PAL) catalyzes the first step in phenylpropanoid metabolism and plays a central role in the biosynthesis of phenylpropanoid compounds. We have previously cloned two PAL genes, PALI and PAL2, from a Populus trichocarpa x P. deltoides F1 hybrid. Here, we describe the properties of PALI and PAL2 promoters and their expression patterns in transgenic tobacco and poplar. The promoters were 75% identical in the regions sequenced, and each contained two copies of AC-rich putative cis-acting elements that matched a consensus plant myb transcription factor binding site sequence. In transgenic tobacco, PALI-GUS and PAL2-GUS fusions directed similar patterns of expression in developing primary xylem of leaves, stems, and other organs, and in secondary xylem of stems. Contrary to previously documented patterns of PAL1/2 expression in poplar, no expression of either fusion was detected in epidermal or subepidermal cell layers of young tobacco leaves or stems. In poplar, the PAL2-GUS fusion directed the highest levels of expression in roots and young leaves and stems. In young leaves and stems, high GUS activity was detected in epidermal or subepidermal cells as well as in primary xylem and phloem fibers. GUS activity was low in woody stems, and was weak or absent in developing secondary xylem. The patterns of PAL2-GUS expression in poplar are very similar to those of PAL1/2 mRNA accumulation in poplar. However, the distinct patterns of expression directed by the PAL2 promoter in poplar and tobacco show that PAL2-GUS expression in tobacco does not accurately reflect all aspects of PAL2 expression in poplar.

The Structure and Cytochemistry of the Stigma-style Complex ofCorylus avellanaL. ‘Tonda Gentile delle Langhe’ (Corylaceae)

Structural and cytochemical aspects of the stigma-style complex ofCorylus avellanawere studied. In cross section the stigmatic style consists of papillae, one or two layers of sub-epidermal cells and a central transmitting tissue. The papillae cover the style for about 80% of its length, are unicellular and are coated with a cuticle-pellicle. During development, the cell walls of the papillae increase in thickness and between them, below the cuticle, lipid bodies are observed. The sub-epidermal cells are similar in cell content to the papillae. The central transmitting tissue consists of highly vacuolated cells and the intercellular spaces are filled with a proteic and polysaccharidic substance. Both the transverse and longitudinal walls contain plasmodesmata.

A morphological and histological comparison of the initiation and development of pecan (Carya illinoinensis) somatic embryogenic cultures induced with naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid

Somatic embryos produced in vitro may exhibit structural abnormalities that affect their subsequent germination and conversion into plants. To assess the influence of auxin type on embryo initiation and development, a morphological and histological comparison was made of pecan (Carya illinoinensis) somatic embryogenic cultures induced on media with naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid (2,4-D), using light and scanning electron microscopy. Both auxins promoted enhanced cell division, particularly in subepidermal cell layers. However, notable differences were observed in mitotic activity, location of embryogenic cell proliferation, epidermal continuity, callus growth, and embryo morphology. Cultures induced on naphthaleneacetic acid had embryogenic regions composed of homogeneous, isodiametric, meristematic cells. Embryos derived from these cultures generally had a normal morphology, were single, and had a discrete apical meristem. In contrast, tissues induced on media with 2,4-D had more intense and heterogeneous regions of cell division. Proliferating cell regions were composed of meristematic cells interspersed with callus and involved more extensive regions of the mesophyll. Marked callus proliferation caused epidermal rupture in some areas. Embryos induced on medium with 2,4-D had a higher incidence of abnormalities that included fasciated, fan-shaped, and tubular embryos. Defined apical meristems were often lacking or partially obliterated due to callus proliferation. The heterogeneous, often intensive proliferation of cells in cultures induced with 2,4-D may interfere with normal patterns of embryo development.

Mutants of Arabidopsis as tools to understand the regulation of phenylpropanoid pathway and UVB protection mechanisms.

Plants accumulate certain phenylpropanoid compounds in the vacuoles of their epidermal and subepidermal cell layers thereby protecting the underlying tissue against UVB-induced damage. However, a number of mutants of Arabidopsis thaliana are known that fail to synthesize these protective pigments, thereby allowing harmful UVB radiation to penetrate into their dermal layers. Study of several of these nonlethal mutants, defective in various aspects of flavonoid and lignin biosynthesis, has led to a better understanding of the coordinate regulation and expression of important genes as well as of mechanisms involved in plant defense against UVB radiation. The characteristics of the various phenylpropanoid mutants of Arabidopsis, viz. flavonoid mutants (banyuls [ban]; increased chalcone synthase expression 1 [icx1]; transparent testa [tt] and ultraviolet sensitive [uvs]) and hydroxycinnamic acid ester mutants (ferulic acid hydroxylase 1 [fah1] and sinapoylglucose accumulator 1 [sng1]) are discussed in detail. We have briefly touched upon, wherever relevant, the unique aspects in other plant species too.

The Mechanics of the Grass Flower: The Extension of the Staminal Filaments and the Lodicules of Maize

In this paper on the flower mechanics of the grasses, the morphological and cytological processes leading to flower opening and anther exsertion are studied. At anthesis synchronous cell extension in both the lodicules and the filaments open the flower and exsert the anthers, respectively. In the lodicules all the cells extend, except the vascular tissues. In the filaments extension is limited to the epidermis and one or two subepidermal cell layers, whereas the more centrally located parenchyma and the vascular tissues are disrupted. Ultrastructural aspects of the rapid vacuolations, necessary for these extensions, are studied using comparisons between TEM and high-resolution SEM.

Adventitious shoot formation in tulip: histological analysis and response to selective agents

On explants taken from young floral stems of tulip ( Tulipa gesneriana L.) adventitious shoot formation was induced. The shoots were formed directly from the explants without an intermediate callus phase. More shoots developed on explants taken from dry-stored bulbs than from bulbs stored in ice. At the basis of the shoots a meristem was formed that developed into a new bulb. The ability to form this bulb meristem was compared in 8 cultivars, and was highest in Lucky Strike and Monte Carlo. Adventitious shoot formation was initiated in the first two subepidermal cell layers and a number of cells of the original explant contributed to the shoot formation. To avoid the formation of chimeric transformed shoots in future transformation, an optimal selection system was established. For selection purposes, the aminoglycoside antibiotics were not very effective in inhibiting shoot formation, since tulip tissue showed a high tolerance for kanamycin, while G418 and hygromycin induced severe necrosis of the explants at low concentrations. In contrast, the herbicides phosphinothricin and glyphosate were very effective and offer good perspectives to be used for selection of transformed shoots.

Characterization of a gene transcribed in the L2 and L3 layers of the tobacco shoot apical meristem.

Recent genetic and molecular studies have been initiated to uncover the fundamental cellular processes unique to the shoot apical meristem. Our previous work resulted in the isolation of a cDNA clone derived from tobacco apex RNA, A3, that appeared to be transcriptionally restricted to the shoot apex regardless of developmental stage. Here the DNA sequence and in situ RNA analysis of A3 is presented. The A3 gene potentially encodes a small hydrophilic polypeptide, the sequence of which is unique to current data bases. It has been found that transcripts of the A3 gene are confined to the subepidermal and internal cell layers of the tobacco shoot apical meristem throughout development, become localized to undifferentiated floral organ primordia, and diminish as the developmental potential of the meristematic tissue becomes restricted.

High level histone H2A gene expression during early stages of adventitious bud formation in Norway spruce (Picea abies)

We have demonstrated the correlation between cell division and the expression of a histone H2A‐encoding gene, His2A, in Norway spruce. Picea abies (L.) Karst and used a cDNA clone in in situ hybridization experiments to monitor the cytokinin‐induced cell division during early stages of adventitious bud development. A general stimulation of division of epidermal and cortical cells followed upon the cytokinin treatment. After two weeks in culture a high mitotic activity was detected only in single cells or small groups of cells in the epidermis and subepidermal cell layers. These cells presumably constitute the early stages of meristem primordia. The small clusters of dividing cells enlarge and subsequently form adventitious buds. Cells of the meristem and needle primordia of adventitious buds divide frequently as do the corresponding cells in vegetative buds. A quiescent center is distinguished within the apical meristem of vegetative buds. These cells, in the summit of the domed meristem, divide with a considerably lower frequency than cells in the flanking region. Differences in the temporal expression pattern of the histone H2A gene in cells of the vascular tissue, detected between embryos germinating in vitro and bud‐induced embryos, suggest that the cytokinin treatment affects the timing of cell divisions in the differentiating procambium.

Molecular and biochemical markers for embryogenic potential and regenerative capacity of barley ( Hordeum vulgare L.) cell cultures

The embryogenic potential and regenerative capacity of cereal cell cultures was examined using the expression of two embryo-specific genes (the dormancy-related cDNA-clone B15C, R. Aalen, Norway; the aldose reductase cDNA-clone pG22–69, D. Bartels, Germany) in barley cell cultures. By Northern blotting the expression of both clones was limited to embryogenic cultures and not to non-embryogenic cultures. In situ hybridization showed high gene expression in the shoot and root apex of embryos and in subepidermal cell layers of embryogenic barley cultures. In androgenetic embryos the expression of the embryo-specific clones was detected as early as 2 weeks after isolation of microspores, indicating their usefulness as early markers for embryogenesis. Nevertheless, cell aggregates which were embryogenic but no longer able to regenerate plants, expressed both genes. Cell cultures consisting of embryogenic aggregates unable to regenerate plantlets were used to verify that an embryo-specific marker alone is not sufficient to characterize regenerable cultures. Comparing the protein pattern of embryogenic/regenerable and embryogenic/non-regenerable suspension cultures of barley, we identified a 85-kDa polypeptide (pI 5.8) accumulating only in non-regenerable cultures. Moreover, following the electrophoretic pattern of secreted proteins, two glycoproteins were found correlating with the embryogenic capacity (46 kDa, pI 6.1) and the loss of regenerative potential (17.4 kDa), respectively. The data indicate that at least two levels of control exist in somatic embryogenesis. One is the induction of bipolar embryos and the second the germination of somatic embryos into plantlets.

Histological Observation of Multiple Shoot Formation Site in Rice Seed Culture.

High cytokinin concentration medium induced multiple shoot. on thc basal part of the main stem generat.ed from cultured rice seeds. Hist:olog'ical observat.ions were done on sections of the basal part of main stem and showcd that multiple shoot originated from two types of bud - axillary buds from the primordium of tiller of the first leaf nodeand adventitious buds from the basal region of the first leaf. The adventitious buds were derived from epidermal and subepidermal cell layers on two fixed sites in the abaxial side of the base of the first leaf, where between these two sites the tiller of tho first leaf node could be found.

Characterization and in situ localization of a salt-induced tomato peroxidase mRNA

NaCl treatment of tomato plants in hydroponic culture at concentrations as low as 50 mM resulted in enhanced accumulation of transcripts of TPX1, a full-length cDNA clone that we had isolated from a library of NaCl-treated tomato plants using a peroxidase-specific oligonucleotide probe. Although the overall amino acid sequence identity of TPX1 to other peroxidase genes was less than 45%, there was a very high degree of identity in all of the conserved domains. The deduced amino acid sequence included the presence of a N-terminal signal peptide but not the C-terminal extension present in peroxidases targeted to the vacuole. The mature protein has a theoretical pI value of 7.5. Transcripts that hybridized to TPX1 were detected only in the roots with higher levels of mRNA in epidermal and subepidermal cell layers. Isoelectric focusing of root extracts showed two major bands of peroxidase activity at pI 5.9 and 6.2. Both activities increased with salt treatment. Southern analysis indicated the presence of only a single TPX1 gene in tomato.

High-frequency Organogenesis from Direct Seed Culture in Lathyrus

Culture conditions were developed for inducing a high frequency of direct shoot morphogenesis and whole plant regeneration in cultures of intact seedlings of Lathyrus cicera L., L. ochrus (L.) DC., L. sativus L., and L. tingitanus L. The procedure of shoot regeneration involved culturing of whole, mature seeds on MS medium containing cytokinins, or thidiazuron (TDZ), a substituted phenylurea with cytokinin-like activity. Differentiation of shoots occurred without an intervening callus phase from the cotyledonary node and surrounding tissues of the intact seedlings developed from seeds germinated on media containing kinetin, BAP or TDZ. An average of 19·0, 15·8, 28·8 and 43·0 shoots were regenerated at optimal concentrations of 50·0, 50·0 and 80·0 μM BAP in L. cicera, L. tingitanus, L. ochrus and L. sativus, respectively. TDZ enhanced the shoot formation at the concentration of 10 μM to an average of 33·1 and 33·7 shoots per seedling in L. cicera and L. tingitanus and at 50 μM, to 57·4 shoots per seedling in L. sativus. Regenerated shoots developed roots on a modified MS medium containing 2·5 μM NAA and the surviving plantlets were transferred to soil. Histological studies revealed de novo formation of shoot buds from the epidermal or subepidermal cells of the basal and nodal region of multiple epicotyls. Several meristematic centres consisting of actively-dividing cells developed in the subepidermal cell layer of the nodal tissue and adjacent areas within 7 d of seed culture on a cytokinin- or TDZ-supplemented medium. The pattern of the development of these meristematic centres and shoot development was similar in all four species.

Development of the olfactory organ in the zebrafish, Brachydanio rerio

The development of the olfactory organ of the zebrafish, from the forming of early placode to the adult organ, was investigated by electron microscopy and DiI labeling. The olfactory placode is formed by a subepidermal layer of cells. These cells differ from those of the epidermis as well as from brain cells, and they do not mingle either with epidermal or with brain cells. No migration of cells from the brain or the epidermis towards the subepidermal cell layer has been observed. The cells of the subepidermal layer seem to form all cell types of the olfactory mucosa, i.e., basal cells, ciliated and microvillous receptor cells, supporting cells, and ciliated nonsensory cells. Axons grow into the forebrain at a very early stage when the epidermis still covers the placode completely. Dendrites grow out when the epidermis separates, building the olfactory pit. This process implicates neither cell lysis nor cell degeneration. The olfactory pit forms a rosette with a midline raphe and olfactory lamellae. The incurrent nostril is separated from the excurrent nostril by a funnel-shaped structure. Differentiation of the olfactory placode in the embryo is accomplished very quickly, whereas the development into the adult organ during larval stages is a slow process.