Carbohydrates in Individual Cells of Epidermis, Mesophyll, and Bundle Sheath in Barley Leaves with Changed Export or Photosynthetic Rate1

Carbohydrate metabolism of barley (Hordeum vulgare) leaves induced to accumulate sucrose (Suc) and fructans was investigated at the single-cell level using single-cell sampling and analysis. Cooling of the root and shoot apical meristem of barley plants led to the accumulation of Suc and fructan in leaf tissue. Suc and fructan accumulated in both mesophyll and parenchymatous bundle-sheath (PBS) cells because of the reduced export of sugars from leaves under cooling and to increased photosynthesis under high photon fluence rates. The general trends of Suc and fructan accumulation were similar for mesophyll and PBS cells. The fructan-to-Suc ratio was higher for PBS cells than for mesophyll cells, suggesting that the threshold Suc concentration needed for the initiation of fructan synthesis was lower for PBS cells. Epidermal cells contained very low concentrations of sugar throughout the cooling experiment. The difference in Suc concentration between control and treated plants was much less if compared at the single-cell level rather than the whole-tissue level, suggesting that the vascular tissue contains a significant proportion of total leaf Suc. We discuss the importance of analyzing complex tissues at the resolution of individual cells to assign molecular mechanisms to phenomena observed at the whole-plant level.

A Fiberless Seed Mutation in Cotton Is Associated with Lack of Fiber Cell Initiation in Ovule Epidermis and Alterations in Sucrose Synthase Expression and Carbon Partitioning in Developing Seeds1

Fiber cell initiation in the epidermal cells of cotton (Gossypium hirsutum L.) ovules represents a unique example of trichome development in higher plants. Little is known about the molecular and metabolic mechanisms controlling this process. Here we report a comparative analysis of a fiberless seed (fls) mutant (lacking fibers) and a normal (FLS) mutant to better understand the initial cytological events in fiber development and to analyze the metabolic changes that are associated with the loss of a major sink for sucrose during cellulose biosynthesis in the mutant seeds. On the day of anthesis (0 DAA), the mutant ovular epidermal cells lacked the typical bud-like projections that are seen in FLS ovules and are required for commitment to the fiber development pathway. Cell-specific gene expression analyses at 0 DAA showed that sucrose synthase (SuSy) RNA and protein were undetectable in fls ovules but were in abundant, steady-state levels in initiating fiber cells of the FLS ovules. Tissue-level analyses of developing seeds 15 to 35 DAA revealed an altered temporal pattern of SuSy expression in the mutant relative to the normal genotype. Whether the altered programming of SuSy expression is the cause or the result of the mutation is unknown. The developing seeds of the fls mutant have also shown several correlated changes that represent altered carbon partitioning in seed coats and cotyledons as compared with the FLS genotype.

A Common Position-Dependent Mechanism Controls Cell-Type Patterning and GLABRA2 Regulation in the Root and Hypocotyl Epidermis of Arabidopsis1

A position-dependent pattern of epidermal cell types is produced during root development in Arabidopsis thaliana. This pattern is reflected in the expression pattern of GLABRA2 (GL2), a homeobox gene that regulates cell differentiation in the root epidermis. GL2 promoter::GUS fusions were used to show that the TTG gene, a regulator of root epidermis development, is necessary for maximal GL2 activity but is not required for the pattern of GL2 expression. Furthermore, GL2-promoter activity is influenced by expression of the myc-like maize R gene (35S::R) in Arabidopsis but is not affected by gl2 mutations. A position-dependent pattern of cell differentiation and GL2-promoter activity was also discovered in the hypocotyl epidermis that was analogous to the pattern in the root. Non-GL2-expressing cell files in the hypocotyl epidermis located outside anticlinal cortical cell walls exhibit reduced cell length and form stomata. Like the root, the hypocotyl GL2 activity was shown to be influenced by ttg and 35S::R but not by gl2. The parallel pattern of cell differentiation in the root and hypocotyl indicates that TTG and GL2 participate in a common position-dependent mechanism to control cell-type patterning throughout the apical-basal axis of the Arabidopsis seedling.

Clues to epidermal cancer proneness revealed by reconstruction of DNA repair-deficient xeroderma pigmentosum skin in vitro

Sun exposure has been clearly implicated in premature skin aging and neoplastic development. These features are exacerbated in patients with xeroderma pigmentosum (XP), a hereditary disease, the biochemical hallmark of which is a severe deficiency in the nucleotide excision repair of UV-induced DNA lesions. To develop an organotypic model of DNA repair deficiency, we have cultured several strains of primary XP keratinocytes and XP fibroblasts from skin biopsies of XP patients. XP skin comprising both a full-thickness epidermis and a dermal equivalent was succesfully reconstructed in vitro. Satisfactory features of stratification were obtained, but the expression of epidermal differentiation products, such as keratin K10 and loricrin, was delayed and reduced. In addition, the proliferation of XP keratinocytes was more rapid than that of normal keratinocytes. Moreover, increased deposition of cell attachment proteins, α-6 and β-1 integrins, was observed in the basement membrane zone, and β-1 integrin subunit, the expression of which is normally confined to basal keratinocytes, extended into several suprabasal cell layers. Most strikingly, the in vitro reconstructed XP skin displayed numerous proliferative epidermal invasions within dermal equivalents. Epidermal invasion and higher proliferation rate are reminiscent of early steps of neoplasia. Compared with normal skin, the DNA repair deficiency of in vitro reconstructed XP skin was documented by long-lasting persistence of UVB-induced DNA damage in all epidermal layers, including the basal layer from which carcinoma develops. The availability of in vitro reconstructed XP skin provides opportunities for research in the fields of photoaging, photocarcinogenesis, and tissue therapy.

Formation of a Normal Epidermis Supported by Increased Stability of Keratins 5 and 14 in Keratin 10 Null Mice

The expression of distinct keratin pairs during epidermal differentiation is assumed to fulfill specific and essential cytoskeletal functions. This is supported by a great variety of genodermatoses exhibiting tissue fragility because of keratin mutations. Here, we show that the loss of K10, the most prominent epidermal protein, allowed the formation of a normal epidermis in neonatal mice without signs of fragility or wound-healing response. However, there were profound changes in the composition of suprabasal keratin filaments. K5/14 persisted suprabasally at elevated protein levels, whereas their mRNAs remained restricted to the basal keratinocytes. This indicated a novel mechanism regulating keratin turnover. Moreover, the amount of K1 was reduced. In the absence of its natural partner we observed the formation of a minor amount of novel K1/14/15 filaments as revealed by immunogold electron microscopy. We suggest that these changes maintained epidermal integrity. Furthermore, suprabasal keratinocytes contained larger keratohyalin granules similar to our previous K10T mice. A comparison of profilaggrin processing in K10T and K10−/− mice revealed an accumulation of filaggrin precursors in the former but not in the latter, suggesting a requirement of intact keratin filaments for the processing. The mild phenotype of K10−/− mice suggests that there is a considerable redundancy in the keratin gene family.

Distinct desmocollin isoforms occur in the same desmosomes and show reciprocally graded distributions in bovine nasal epidermis.

The adhesive core of the desmosome is composed of cadherin-like glycoproteins of two families, desmocollins and desmogleins. Three isoforms of each are expressed in a tissue-specific and developmentally regulated pattern. In bovine nasal epidermis, the three desmocollin (Dsc) isoforms are expressed in overlapping domains; Dsc3 expression is strongest in the basal layer, while Dsc2 and Dsc1 are strongly expressed in the suprabasal layers. Herein we have investigated whether different isoforms are assembled into the same or distinct desmosomes by performing double immunogold labeling using isoform-specific antibodies directed against Dsc1 and Dsc3. The results show that individual desmosomes harbor both isoforms in regions where their expression territories overlap. Quantification showed that the ratio of the proteins in each desmosome altered gradually from basal to immediately suprabasal and upper suprabasal layers, labeling for Dsc1 increasing and Dsc3 decreasing. Thus desmosomes are constantly modified as cells move up the epidermis, with continuing turnover of the desmosomal glycoproteins. Statistical analysis of the quantitative data showed a possible relationship between the distributions of the two isoforms. This gradual change in desmosomal composition may constitute a vertical adhesive gradient within the epidermis, having important consequences for cell positioning and differentiation.

Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes.

Cellular senescence is defined by the limited proliferative capacity of normal cultured cells. Immortal cells overcome this regulation and proliferate indefinitively. One step in the immortalization process may be reactivation of telomerase activity, a ribonucleoprotein complex, which, by de novo synthesized telomeric TTAGGG repeats, can prevent shortening of the telomeres. Here we show that immortal human skin keratinocytes, irrespective of whether they were immortalized by simian virus 40, human papillomavirus 16, or spontaneously, as well as cell lines established from human skin squamous cell carcinomas exhibit telomerase activity. Unexpectedly, four of nine samples of intact human skin also were telomerase positive. By dissecting the skin we could show that the dermis and cultured dermal fibroblasts were telomerase negative. The epidermis and cultured skin keratinocytes, however, reproducibly exhibited enzyme activity. By separating different cell layers of the epidermis this telomerase activity could be assigned to the proliferative basal cells. Thus, in addition to hematopoietic cells, the epidermis, another example of a permanently regenerating human tissue, provides a further exception of the hypothesis that all normal human somatic tissues are telomerase deficient. Instead, these data suggest that in addition to contributing to the permanent proliferation capacity of immortal and tumor-derived keratinocytes, telomerase activity may also play a similar role in the lifetime regenerative capacity of normal epidermis in vivo.

Inflammatory skin disease in transgenic mice that express high levels of interleukin 1 alpha in basal epidermis.

Resting epidermal keratinocytes contain large amounts of interleukin 1 (IL-1), but the function of this cytokine in the skin remains unclear. To further define the role of IL-1 in cutaneous biology, we have generated two lines of transgenic mice (TgIL-1.1 and TgIL-1.2) which overexpress IL-1 alpha in basal keratinocytes. There was high-level tissue-specific expression of transgene mRNA and protein and large quantities of IL-1 alpha were liberated into the circulation from epidermis in both lines. TgIL-1.1 mice, which had the highest level of transgene expression, developed a spontaneous skin disease characterized by hair loss, scaling, and focal inflammatory skin lesions. Histologically, nonlesional skin of these animals was characterized by hyperkeratosis and a dermal mononuclear cell infiltrate of macrophage/monocyte lineage. Inflammatory lesions were marked by a mixed cellular infiltrate, acanthosis, and, in some cases, parakeratosis. These findings confirm the concept of IL-1 as a primary cytokine, release of which is able to initiate and localize an inflammatory reaction. Furthermore, these mice provide the first definitive evidence that inflammatory mediators can be released from the epidermis to enter the systemic circulation and thereby influence, in a paracrine or endocrine fashion, a wide variety of other cell types.

Correlation of individual papilloma latency time with DNA adducts, 8-hydroxy-2'-deoxyguanosine, and the rate of DNA synthesis in the epidermis of mice treated with 7,12-dimethylbenz[alpha]anthracene.

The question was addressed whether the risk of cancer of an individual in a heterogeneous population can be predicted on the basis of measurable biochemical and biological variables postulated to be associated with the process of chemical carcinogenesis. Using the skin tumor model with outbred male NMRI mice, the latency time for the appearance of a papilloma was used as an indicator of the individual cancer risk. Starting at 8 weeks of age, a group of 29 mice was treated twice weekly with 20 nmol of 7,12-dimethylbenz[alpha]anthracene (DMBA) applied to back skin. The individual papilloma latency time ranged from 13.5 to 25 weeks of treatment. Two weeks after the appearance of the first papilloma in each mouse, an osmotic minipump delivering 5-bromo-2'-deoxyuridine was s.c. implanted and the mouse was killed 24 hr later. Levels of DMBA-DNA adducts, of 8-hydroxy-2'-deoxyguanosine, and various measures of the kinetics of cell division were determined in the epidermis of the treated skin area. The levels of 8-hydroxy-2'-deoxyguanosine and the fraction of cells in DNA replication (labeling index for the incorporation of 5-bromo-2'-deoxyuridine) were significantly higher in those mice that showed short latency times. On the other hand, the levels of DMBA-DNA adducts were lowest in animals with short latency times. The latter finding was rather unexpected but can be explained as a consequence of the inverse correlation seen for the labeling index: with each round of cell division, the adduct concentration is reduced to 50% because the new DNA strand is free of DMBA adducts until the next treatment. Under the conditions of this bioassay, therefore, oxygen radical-related genotoxicity and the rate of cell division, rather than levels of carcinogen-DNA adducts, were found to be of predictive value as indicators of an individual cancer risk.

A Xenopus distal-less gene in transgenic mice: conserved regulation in distal limb epidermis and other sites of epithelial-mesenchymal interaction.

In this paper, we show the conserved regulation of the homeodomain gene Distal-less-3 (Dlx-3) by analyzing the expression of a promoter from the Xenopus ortholog, Xdll-2, in transgenic mice. A 470-bp frog regulatory sequence confers appropriate expression on a lacZ reporter gene in the ectodermal component of structures derived from epithelial-mesenchymal interactions. Remarkably, this includes structures absent in Xenopus, such as the hair follicle and mammary gland, suggesting that conserved regulatory elements can be used to control the formation of structures peculiar to individual species. In addition, expression of Dlx-3 in developing limbs is highest at the most distal portion. This pattern is duplicated by the Xenopus promoter, indicating that this DNA may include sequences responsive to conserved proximodistal patterning signals in the vertebrate limb.

Geodynamic events reconstructed in the Betic, Maghrebian, and Apennine chains (central-western Tethys)

A synthetic study has been made to identify main tectono-sedimentary and geodynamic events in central-western Tethys. For this, an interdisciplinary analysis has been performed on successions belonging to tectonic units derived from Betic-Maghrebian-southern Apennine “Flysch basin” domain. The stratigraphic records of the internal, external, and mixed successions deposited in lateral basins of different chains show very similar characters, especially regarding: (a) lithostratigraphy and ages; (b) kind and provenance of supplies (immature and supermature petrofacies from internal and external margins, respectively); (c) presence of “mixed successions” (composed of alternating internal and external petrofacies) attesting to clear palaeogeographic relationships between opposite depositional systems; and (d) timing of the deformation. In addition, specific lithofacies reveal correspondence with similar sedimentary events, such as: (1) very thick silicoclastic supply concentrated in restricted time ranges indicating the main deformational phases in the margin/basin systems; (2) levels rich in black-shales, glauconian, siliceous-producers, and volcaniclastic intercalations, widespread in the studied successions and linked to particular events at the Tethyan scale (anoxic periods, starvation-upwelling, acid-intermediate penecontemporaneous volcanic activity, respectively). Tectonic influence has also been recorded by means of unconformities and tectofacies (such as turbidites, slumps, or olisthostromes, etc.), being correlated with the main deformational phases. Similar stratigraphic and tectonic events have also been found in the Calvana unit of Val Marecchia nappe (Ligurides, northern Apennine). Correlations of stratigraphic and tectonic events support the proposal of an evolutionary geodynamic model involving the presence of a “Mesomediterranean microplate” in intermediate position during Africa-Europe convergence. The closure of central-western Tethys occasioned the Betic-Maghrebian-southern Apennine oceanic branch deformation and the birth of perimediterranean chains during middle-late Miocene.

(Table S1) Boron isotope and trace element data and reconstructed carbonate system parameters, ODP Holes 122-761B and 154-926A

The middle Miocene Climatic Optimum (17-15 Ma; MCO) is a period of global warmth and relatively high CO2 and is thought to be associated with a significant retreat of the Antarctic Ice Sheet (AIS). We present here a new planktic foraminiferal d11B record from 16.6 to 11.8 Ma from two deep ocean sites currently in equilibrium with the atmosphere with respect to CO2. These new data demonstrate that the evolution of global climate during the middle Miocene (as reflected by changes in the cyrosphere) was well correlated to variations in the concentration of atmospheric CO2. What is more, within our sampling resolution (~1 sample per 300 kyr) there is no evidence of hysteresis in the response of ice volume to CO2 forcing during the middle Miocene, contrary to what is understood about the Antarctic Ice Sheet from ice sheet modelling studies. In agreement with previous data, we show that absolute levels of CO2 during the MCO were relatively modest (350-400 ppm) and levels either side of the MCO are similar or lower than the pre-industrial (200-260 ppm). These new data imply the presence of either a very dynamic AIS at relatively low CO2 during the middle Miocene or the advance and retreat of significant northern hemisphere ice. Recent drilling on the Antarctic margin and shore based studies indicate significant retreat and advance beyond the modern limits of the AIS did occur during the middle Miocene, but the complete loss of the AIS was unlikely. Consequently, it seems that ice volume and climate variations during the middle Miocene probably involved a more dynamic AIS than the modern but also some component of land-based ice in the northern hemisphere.

Carbonate preservation reconstructed from ODP Sites 154-927 and 154-929 at Ceará Rise

Enhanced Atlantic overturning during the Pliocene was first proposed almost 10 yrs ago. Evidence for this Pliocene super conveyor scenario has been collected using a number of proxies (e.g., benthic d13C, Nd isotopic composition of manganese crusts). The present study contributes to the existing evidences by using carbonate dissolution and current vigour history of early Pliocene sediments from the Ceará Rise (ODP Sites 927 and 929). In order to reveal carbonate dissolution history, a number of commonly used and newly established proxies were applied, i.e., sand and carbonate contents, foraminifer fragmentation index, Bulloides Dissolution Index and carbonate silt grain-size distributions. Terrigenous silt grain-size distributions were used to unravel variations in relative current strength and sediment input to the two sites. Overall good carbonate preservation at the shallow Site 927 (3314 m water depth) shows that this level was bathed in North Atlantic Deep Water throughout the early Pliocene. The contrastingly poor carbonate preservation record of the deeper Site 929 (4358 m water depth, at present exposed to Antarctic Bottom Water) is frequently interrupted by phases of good carbonate preservation. These results indicate that the depth of the calcite lysocline was mainly tied to present level (ab. 4200 m water depth), and sometimes even dropped to water depths greater than 4360 m due to even more enhanced circulation. Surprisingly the expansion of NADW is not clearly reflected by an increase in current speed as shown by continuously fine terrigenous grain size.