Spongy Mesophyll Research Articles

Comparative leaf growth strategies in response to low-water and low-light availability: variation in leaf physiology underlies variation in leaf mass per area in Populus tremuloides.

Developmental phenotypic plasticity can allow plants to buffer the effects of abiotic and biotic environmental stressors. Therefore, it is vital to improve our understanding of how phenotypic plasticity in ecological functional traits is coordinated with variation in physiological performance in plants. To identify coordinated leaf responses to low-water (LW) versus low-light (LL) availability, we measured leaf mass per area (LMA), leaf anatomical characteristics and leaf gas exchange of juvenile Populus tremuloidesMichx. trees. Spongy mesophyll tissue surface area (Asmes/A) was correlated with intrinsic water-use efficiency (WUEi: photosynthesis, (Aarea)/stomatal conductance (gs)). Under LW availability, these changes occurred at the cost of greater leaf tissue density and reduced expansive growth, as leaves were denser but were only 20% the final area of control leaves, resulting in elevated LMA and elevated WUEi. Low light resulted in reduced palisade mesophyll surface area (Apmes/A) while spongy mesophyll surface area was maintained (Asmes/A), with no changes to WUEi. These leaf morphological changes may be a plastic strategy to increase laminar light capture while maintaining WUEi. With reduced density and thickness, however, leaves were 50% the area of control leaves, ultimately resulting in reduced LMA. Our results illustrate that P. tremuloides saplings partially maintain physiological function in response to water and light limitation by inducing developmental plasticity in LMA with underlying anatomical changes. We discuss additional implications of these results in the context of developmental plasticity, growth trade-offs and the ecological impacts of climate change.

Nickel biopathways in tropical nickel hyperaccumulating trees from Sabah (Malaysia)

The extraordinary level of accumulation of nickel (Ni) in hyperaccumulator plants is a consequence of specific metal sequestering and transport mechanisms, and knowledge of these processes is critical for advancing an understanding of transition element metabolic regulation in these plants. The Ni biopathways were elucidated in three plant species, Phyllanthus balgooyi, Phyllanthus securinegioides (Phyllanthaceae) and Rinorea bengalensis (Violaceae), that occur in Sabah (Malaysia) on the Island of Borneo. This study showed that Ni is mainly concentrated in the phloem in roots and stems (up to 16.9% Ni in phloem sap in Phyllanthus balgooyi) in all three species. However, the species differ in their leaves – in P. balgooyi the highest Ni concentration is in the phloem, but in P. securinegioides and R. bengalensis in the epidermis and in the spongy mesophyll (R. bengalensis). The chemical speciation of Ni2+ does not substantially differ between the species nor between the plant tissues and transport fluids, and is unambiguously associated with citrate. This study combines ion microbeam (PIXE and RBS) and metabolomics techniques (GC-MS, LC-MS) with synchrotron methods (XAS) to overcome the drawbacks of the individual techniques to quantitatively determine Ni distribution and Ni2+ chemical speciation in hyperaccumulator plants.

Effects of Bemisia tabaci (Gennadius) infestation and squash silverleaf disorder on Cucurbita pepo L. leaf

The aetiology of squash silverleaf (SSL) induced by the infestation of Bemisia tabaci (Gennadius) ‘biotype’ B (B. tabaci-B) is poorly understood. To help to find out how B. tabaci destroys Cucurbita pepo L. and induces SSL, changes induced by SSL and responses of C. pepo to B. tabaci-B infestation were studied by comparing chlorophyll fluorescence parameters and biochemical, morphological and anatomical aspects of grade 0–4 SSL leaves. The results showed that chlorophyll contents decreased and the chlorophyll a/b ratio increased with the increase of silvered leaf, which indicated that the decrease of chlorophyll contents led to silvered leaf. Anatomical observation showed that B. tabaci-B infestation changed the shape and arrangement of lower epidermis cells and spongy mesophyll cells. Moreover, air spaces appeared between upper epidermis and palisade tissue and in spongy tissue, which might be the cause of the increase in leaf thickness. Additionally, the altering of former spaces light reflection was another reason for leaf silvering. As response to B. tabaci-B infestation, the content of soluble proteins and soluble sugar decreased, and the content of Malondialdehyde (MDA) increased. Pearson correlation showed a significant correlation between fluorescence parameters (except α) and the other parameters measured in this study. The result also suggested that chlorophyll fluorescence can be widely used in field studies as an early diagnostic measure of stress in plant caused by adverse environmental conditions, especially by insect pests.

Leaf anatomy, BVOC emission and CO2 exchange of arctic plants following snow addition and summer warming.

Climate change in the Arctic is projected to increase temperature, precipitation and snowfall. This may alter leaf anatomy and gas exchange either directly or indirectly. Our aim was to assess whether increased snow depth and warming modify leaf anatomy and affect biogenic volatile organic compound (BVOC) emissions and CO2 exchange of the widespread arctic shrubs Betula nana and Empetrum nigrum ssp. hermaphroditum METHODS: Measurements were conducted in a full-factorial field experiment in Central West Greenland, with passive summer warming by open-top chambers and snow addition using snow fences. Leaf anatomy was assessed using light microscopy and scanning electron microscopy. BVOC emissions were measured using a dynamic enclosure system and collection of BVOCs into adsorbent cartridges analysed by gas chromatography-mass spectrometry. Carbon dioxide exchange was measured using an infrared gas analyser. Despite a later snowmelt and reduced photosynthesis for B. nana especially, no apparent delays in the BVOC emissions were observed in response to snow addition. Only a few effects of the treatments were seen for the BVOC emissions, with sesquiterpenes being the most responsive compound group. Snow addition affected leaf anatomy by increasing the glandular trichome density in B. nana and modifying the mesophyll of E. hermaphroditum The open-top chambers thickened the epidermis of B. nana, while increasing the glandular trichome density and reducing the palisade:spongy mesophyll ratio in E. hermaphroditum CONCLUSIONS: Leaf anatomy was modified by both treatments already after the first winter and we suggest links between leaf anatomy, CO2 exchange and BVOC emissions. While warming is likely to reduce soil moisture, melt water from a deeper snow pack alleviates water stress in the early growing season. The study emphasizes the ecological importance of changes in winter precipitation in the Arctic, which can interact with climate-warming effects.

Shading and natural rooting biostimulator enhance potential for vegetative propagation of dogwood plants (Cornus alba L.) via stem cuttings

Auxins have been widely used to stimulate rhizogenesis in stem woody cuttings. Recently, more environmentally friendly biostimulators are reported to enhance rhizogenesis. Also, stock plants shading appears to be an easy and effective method to increase rooting of cuttings. The aim of the study was to examine the effect of shading and exogenous application of auxin or biostimulator AlgaminoPlant on rooting potential of two cultivars of dogwood (Cornus alba L.) and to detect anatomical modifications induced by shading that may be related to rooting ability. Stock plants were shaded with a nursery net reducing 50% of light for 6weeks prior to cuttings' harvest while the control stock plants were grown under ambient light. Cuttings from shaded and non-shaded plants were treated with a commercial rooting powder (1% IBA), sprayed with water solution of IBA (200mg·l−1) or with biostimulator AlgaminoPlant (0.2% v/v), whereas the control cuttings were sprayed with water. The effects of treatments were evaluated after 8weeks of rooting in a plastic tunnel. The two cultivars tested differed in natural as well as induced rooting potential, both in cuttings harvested from shaded stock plants and those grown under ambient light. Shading of stock plants significantly improved rooting abilities in comparison to control cuttings from plants grown under ambient light. Shading reduced diameters of stem and pith, with thinner layers of cortex, epidermis and collenchyma; vascular bundles and diameters of xylem vessels were also smaller. The thickness of leaf blades collected from shaded stock plants was lower due to thinner epidermal as well as spongy and palisade mesophyll layers. The best effects in rooting were obtained when shading was followed by the application of rooting inducers. The effects of synthetic auxin IBA and biostimulator AlgaminoPlant were similar; therefore, the latter is a viable option to reduce widespread use of rooting inducing chemicals based on synthetic auxins.

Functional analysis of a viroid RNA motif mediating cell-to-cell movement in Nicotiana benthamiana.

Cell-to-cell trafficking through different cellular layers is a key process for various RNAs including those of plant viruses and viroids, but the regulatory mechanisms involved are still not fully elucidated and good model systems are important. Here, we analyse the function of a simple RNA motif (termed 'loop19') in potato spindle tuber viroid (PSTVd) which is required for trafficking in Nicotiana benthamiana leaves. Northern blotting, reverse transcriptase PCR (RT-PCR) and in situ hybridization analyses demonstrated that unlike wild-type PSTVd, which was present in the nuclei in all cell types, the trafficking-defective loop19 mutants were visible only in the nuclei of upper epidermal and palisade mesophyll cells, which shows that PSTVd loop19 plays a role in mediating RNA trafficking from palisade to spongy mesophyll cells in N.benthamiana leaves. Our findings and approaches have broad implications for studying the RNA motifs mediating trafficking of RNAs across specific cellular boundaries in other biological systems.

Adaptasi Ekofisiologi Terhadap Iklim Tropis Kering: Studi Anatomi Daun Sepuluh Jenis Tumbuhan Berkayu

ABSTRACT Study on foliar anatomic structure of tropical plants are useful to study various aspects of plant life related to photosynthetic, productivity, carbon sequestration, adaptation to environmental stresses as the results of global climate change, etc. This study was conducted to investigate foliar anatomic structure of ten tropical woody plant species including Barringtonia asiatica, Dracontomelon dao, Heritiera littoralis, Diospyros discolor, Calophyllum inophyllum, Antidesma bunius, Schleichera oleosa, Syzygium cumini, Madhuca longifolia dan Adenanthera pavonina; also to discuss its implication to ecophysiological adaptation to tropical dry climate. The results showed that ten woody plant species studied had variation of adaptation pattern to dry tropical climate, exposure to high light intensity or open shade and photosynthesis activity. Plant species in Group II (Antidesma bunius, Madhuca longifolia and Adenanthera pavonina) has relatively higher adaptation compared to Group I (Diospyros discolor, Calophylum inophyllum, Dracontomelon dao, Schleichera oleosa, Heritiera littoralis, Baringtonia asiatica and Syzygium cumini). Some foliar anatomic structures including palisade shape and size, stomatal index, stomatal density, spongy mesophyll thickness, the ratio of P/T and P/S are important characters that can be used as indicator of changes in environmental factors; particularly the density of stomata as indicators of climate change in dry tropic area. Keywords: adaptation, dry climate, eco-physiology, foliar anatomy, woody plant

Limited acclimation in leaf anatomy to experimental drought in tropical rainforest trees.

Dry periods are predicted to become more frequent and severe in the future in some parts of the tropics, including Amazonia, potentially causing reduced productivity, higher tree mortality and increased emissions of stored carbon. Using a long-term (12 year) through-fall exclusion (TFE) experiment in the tropics, we test the hypothesis that trees produce leaves adapted to cope with higher levels of water stress, by examining the following leaf characteristics: area, thickness, leaf mass per area, vein density, stomatal density, the thickness of palisade mesophyll, spongy mesophyll and both of the epidermal layers, internal cavity volume and the average cell sizes of the palisade and spongy mesophyll. We also test whether differences in leaf anatomy are consistent with observed differential drought-induced mortality responses among taxa, and look for relationships between leaf anatomy, and leaf water relations and gas exchange parameters. Our data show that trees do not produce leaves that are more xeromorphic in response to 12 years of soil moisture deficit. However, the drought treatment did result in increases in the thickness of the adaxial epidermis (TFE: 20.5 ± 1.5 µm, control: 16.7 ± 1.0 µm) and the internal cavity volume (TFE: 2.43 ± 0.50 mm3 cm−2, control: 1.77 ± 0.30 mm3 cm−2). No consistent differences were detected between drought-resistant and drought-sensitive taxa, although interactions occurred between drought-sensitivity status and drought treatment for the palisade mesophyll thickness (P = 0.034) and the cavity volume of the leaves (P = 0.025). The limited response to water deficit probably reflects a tight co-ordination between leaf morphology, water relations and photosynthetic properties. This suggests that there is little plasticity in these aspects of plant anatomy in these taxa, and that phenotypic plasticity in leaf traits may not facilitate the acclimation of Amazonian trees to the predicted future reductions in dry season water availability.

Studies on some anatomical features of selected plant species grown in sand dune areas of North Sinai, Egypt

The present investigation aims at studying some anatomical features of some selected native plant species in arid environments of sand dunes at North Sinai, Egypt. Plant samples were collected from three localities; Coastal sand dunes of Balouza (Zygophyllum album, Zygophyllaceae; root, stem and leaf); sand sheets of El-Arish city (stems of Anabasis articulata, Amaranthaceae and Salsola tetrandra, Chenopodiaceae) and El-Owga interdune areas (Fagonia indica, Zygophyllaceae; stem and root, Zygophyllum album, stem). Anatomical adaptations in leaves of Zygophyllum album include the presence of palisade layers on the adaxial and abaxial surfaces, thick cuticle layer cover the epidermis, spongy mesophyll and the presence of water storage cells. Stem anatomical adaptations in different plant species as general observations were the presence of thick cuticle, multiepidermal layers, palisade shape chlorenchyma, patches of sclerenchymatous fiber in the cortex and the precipitation of raphid and druse crystals of different sizes in plant tissues. Root adaptations in Zygophyllum album and Fagonia indica include the presence of periderm layers composed of compacted and elongated cells of different origins and increase the number of small xylem vessels which can guarantee the continuity of water uptake from soil in the case of xylem embolism.

Acclimation Changes of Flavonoids in Needles of Conifers during Heat and Drought Stress 2015

The long-term harsh climate conditions in 2015 distorted already from June up to November in all study trees of Tsuga and Taxus the intracellular organization of the needles. Intimately involved in these repressive processes were the flavanols, a small subgroup of the flavonoids. They were not only deposited in vacuoles of conifer needles but also in the nuclei and chromosomes. Among the many flavonoids the small group of catechin derivatives and polymers named flavanols can exclusively be stained blue with DMACA (dimethylaminocinnamaldehyde). From mid-July onward, the vacuolar flavanols of the epidermal cell layers were gradually diminished as evidenced by decreasing blue staining of nuclei and vacuoles. Subsequently, in August also the large spongy mesophyll cells showed the flavanols decreasing progressively. Apparently, the antioxidant flavanols operate as oxygen radical scavengers. (ROS) were used up during the harsh environmental stress conditions. Both, Tsuga and Taxus reacted in this way. However, it is quite surprising that in all study trees the palisade cells did not contain such vacuolar flavanols. Only these cells were in June the first to show a loss of chlorophyll from chloroplasts as well as an efflux of flavanols from the nuclei. Conversely, from September onward another group of phenols, the yellow-staining flavanols were newly formed in the palisade cells and later on also in the mesophyll cells. Obviously, they were assembled finally to stabilize finally the fragile cell sites. Summing up, the present study shows by cytological studies that the climatic conditions in 2015 produced the worst disturbance of subcellular structures observed since 2000 when our studies on nuclear phenols in needles of conifers were initiated.

Visualizing heterogeneity of photosynthetic properties of plant leaves with two-photon fluorescence lifetime imaging microscopy

Two-photon fluorescence lifetime imaging microscopy (FLIM) was used to analyse the distribution and properties of Photosystem I (PSI) and Photosystem II (PSII) in palisade and spongy chloroplasts of leaves from the C3 plant Arabidopsis thaliana and the C4 plant Miscanthus x giganteus. This was achieved by separating the time-resolved fluorescence of PSI and PSII in the leaf. It is found that the PSII antenna size is larger on the abaxial side of A. thaliana leaves, presumably because chloroplasts in the spongy mesophyll are “shaded” by the palisade cells. The number of chlorophylls in PSI on the adaxial side of the A. thaliana leaf is slightly higher. The C4 plant M. x giganteus contains both mesophyll and bundle sheath cells, which have a different PSI/PSII ratio. It is shown that the time-resolved fluorescence of bundle sheath and mesophyll cells can be analysed separately. The relative number of chlorophylls, which belong to PSI (as compared to PSII) in the bundle sheath cells is at least 2.5 times higher than in mesophyll cells. FLIM is thus demonstrated to be a useful technique to study the PSI/PSII ratio and PSII antenna size in well-defined regions of plant leaves without having to isolate pigment-protein complexes.

X-ray microtomography provides new insights into vacuum impregnation of spinach leaves

Vacuum impregnation is used in the food industry to facilitate the impregnation of porous products with, e.g. firming, antioxidant, antimicrobial or cryoprotective agents. X-ray micro-tomography (μCT) was used to study the process of vacuum impregnation in spinach leaves. Low (300 mbar absolute pressure) and mild vacuum (150 mbar absolute pressure) impregnation protocols were used to impregnate an isotonic solution of trehalose in the leaves and μCT was used to make observations of the cross section of the impregnated samples and quantify their porosity. Results revealed that the free volume in the spongy mesophyll is easier to impregnate than the spaces around the palisade mesophyll. The low vacuum impregnation protocol provoked less impregnation close to the edge of the leaf than in its centre, probably accounting for an influence of the tissue structure on impregnation. The vacuum impregnation protocols tested in this investigation drastically decreased the proportion of large pores (>100 μm) and increased the proportion of small pores (<50 μm). The mild vacuum impregnation protocol, which was designed on the basis of measured apparent porosity, did not achieve full impregnation of the tissue.

Photosynthetic responses of Chrysanthemum morifolium to growth irradiance: morphology, anatomy and chloroplast ultrastructure

Seedlings of Chrysanthemum, cultivar ‘Puma Sunny’, were grown under a range of shading regimes (natural full sunlight, 55, 25, and 15% of full sunlight) for 18 days. Here, we characterized effects of varying light regimes on plant morphology, photosynthesis, chlorophyll fluorescence, anatomical traits, and chloroplast ultrastructure. We showed that leaf color was yellowish-green under full sunlight. Leaf area, internode length, and petiole length of plants were the largest under 15% irradiance. Net photosynthetic rate, water-use efficiency, PSII quantum efficiency, and starch grain were reduced with decreasing irradiance from 100 to 15%. Heavy shading resulted in the partial closure of PSII reaction centers and the CO2 assimilation was restricted. The results showed the leaves of plants were thinner under 25 and 15% irradiance with loose palisade tissue and irregularly arranged spongy mesophyll cells, while the plants grown under full sunlight showed the most compact leaf palisade parenchyma. Irradiance lesser than 25% of full sunlight reduced carbon assimilation and led to limited plant growth. Approximately 55% irradiance was suggested to be the optimal for Chrysanthemum morifolium.

Plasticity in leaf-level water relations of tropical rainforest trees in response to experimental drought.

Summary The tropics are predicted to become warmer and drier, and understanding the sensitivity of tree species to drought is important for characterizing the risk to forests of climate change. This study makes use of a long‐term drought experiment in the Amazon rainforest to evaluate the role of leaf‐level water relations, leaf anatomy and their plasticity in response to drought in six tree genera.The variables (osmotic potential at full turgor, turgor loss point, capacitance, elastic modulus, relative water content and saturated water content) were compared between seasons and between plots (control and through‐fall exclusion) enabling a comparison between short‐ and long‐term plasticity in traits. Leaf anatomical traits were correlated with water relation parameters to determine whether water relations differed among tissues.The key findings were: osmotic adjustment occurred in response to the long‐term drought treatment; species resistant to drought stress showed less osmotic adjustment than drought‐sensitive species; and water relation traits were correlated with tissue properties, especially the thickness of the abaxial epidermis and the spongy mesophyll.These findings demonstrate that cell‐level water relation traits can acclimate to long‐term water stress, and highlight the limitations of extrapolating the results of short‐term studies to temporal scales associated with climate change.

Comparative Leaves Transcriptome Analysis Emphasizing on Accumulation of Anthocyanins in Brassica: Molecular Regulation and Potential Interaction with Photosynthesis.

The purple leaf pigmentation mainly associated with anthocyanins accumulation is common in Brassica but the mechanisms of its production and its potential physiological functions are poorly understood. Here, we performed the phenotypic, cytological, physiological, and comparative leaves transcriptome analyses of 11 different varieties belonging to five Brassica species with purple or green leaves. We observed that the anthocyanin was accumulated in most of vegetative tissues in all species and also in reproduction organs of B. carinata. Anthocyanin accumulated in different part of purple leaves including adaxial and abaxial epidermal cells as well as palisade and spongy mesophyll cells. Leave transcriptome analysis showed that almost all late biosynthetic genes (LBGs) of anthocyanin, especially Dihydroflavonol 4-Reductase (DFR), Anthocyanidin Synthase (ANS) and Transparent Testa 19 (TT19), were highly up-regulated in all purple leaves. However, only one of transcript factors in anthocyanin biosynthesis pathway, Transparent Testa 8 (TT8), was up regulated along with those genes in all purple leaves, indicating its pivotal role for anthocyanin production in Brassica. Interestingly, with the up-regulation of genes for anthocyanin synthesis, Cytosolic 6-phosphogluconolactonase (PLG5) which involved in the oxidative pentose-phosphate pathway was up-regulated in all purple leaves and three genes FTSH PROTEASE 8 (FTS8), GLYCOLATE OXIDASE 1 (GOX1), and GLUTAMINE SYNTHETASE 1;4 (GLN1;4) related to degradation of photo-damaged proteins in photosystem II and light respiration were down-regulated. These results highlighted the potential physiological functions of anthocyanin accumulation related to photosynthesis which might be of great worth in future.

Three-Dimensional Imaging of Plant Organs Using a Simple and Rapid Transparency Technique.

Clearing techniques eliminate factors that interfere with microscopic observation, including light scattering and absorption by pigments and cytoplasmic components. The techniques allow fluorescence-based detailed analyses of materials and characterization of the three-dimensional structure of organs. We describe a simple and rapid clearing and imaging method, termed 'TOMEI' (Transparent plant Organ MEthod for Imaging), which enables microscopic observation of intact plant organs. This method involves a clearing reagent containing 2,2'-thiodiethanol. Conveniently, transparent plant organs were prepared within only 3-6 h. We detected fluorescent stains at a depth of approximately 200 µm using confocal laser scanning microscopy and analyzed fluorescent proteins in internal tissues of transparent organs cleared using TOMEI. We adapted TOMEI for various plant organs of Arabidopsis thaliana and Oryza sativa, including leaves, flower buds, flower stalks, root and nematode-infected root-knots. We visualized whole leaves of A. thaliana from the adaxial epidermis to the abaxial epidermis as well as protoxylem and metaxylem vessels of vascular bundles embedded in spongy mesophyll cells. Inner floral organs were observed in flower buds cleared using TOMEI without the need to prepare sections or remove sepals. Multicolor imaging of fluorescent proteins and dyes, and analyses of the three-dimensional structure of plant organs based on optical sections are possible using TOMEI. We analyzed root-knots cleared using TOMEI and revealed that nematodes induce giant cell expansion in a DNA content-dependent manner. The TOMEI method is applicable to analysis of fluorescent proteins and dyes quantitatively with cell morphological characteristics in whole plant organs.

Virtual microstructural leaf tissue generation based on cell growth modeling

A cell growth algorithm for virtual leaf tissue generation is presented based on the biomechanics of plant cells in tissues. The algorithm can account for typical differences in epidermal layers, palisade mesophyll layer and spongy mesophyll layer which have characteristic differences in the shape of cells, arrangement of cells and void fractions present in each layer. The cell is considered as a closed thin walled structure, maintained in tension by turgor pressure. The cell walls are modelled as linear elastic elements which obey Hooke's law. A Voronoi tessellation was used to generate the initial topology of the cells in the spongy mesophyll layer. Then two layers of brick structured cells are added to the top of it to represent the palisade mesophyll and upper epidermis and a single layer is added at bottom of the Voronoi tessellation to represent the lower epidermal layer. Intercellular air spaces are generated by separating the Voronoi cells along the edges starting from where three Voronoi cells are in contact (schizogenous origin) and/or by deleting some of the Voronoi cells (lysigenous origin). Cell expansion then results from turgor pressure acting on the yielding cell wall material. To find the sequence of positions of each vertex and thus the shape of the tissue with time, a system of differential equations for the positions and velocities of each vertex is established and solved using the ordinary differential equation solver in MatLab. Statistical comparison of the cellular characteristics with 2D cross-sectional slices of real leaf tissue of tomato is excellent. The virtual tissues can be used to systematically study effects of leaf structure on water and gas exchange.

Leaf Blade Anatomy of Philippine Medinilla Gaudich. Species in Relation to their Epiphytic Habit

Leaf anatomy of four epiphytic species of Philippine Medinilla , namely, M. magnifica Lindl., M. miniata Merr., M. pendula Merr., and M. teysmannii Miq., was characterized by observation of leaf cross sections prepared through paraffin method. Similarities among the four species revealed by this study include thick hypodermis below the upper epidermis, palisade mesophyll comprising one layer of elongated lignified parenchyma cells, spongy mesophyll with scarce air spaces, and polocytic stomates present only in the lower epidermis. These features aid in water conservation which is highly crucial among epiphytes. Presence of druse crystals in all four species was observed. This feature functions for structural support, defense against herbivory, calcium storage, and increasing photosynthetic efficiency. In hypodermis, number of cell layers, cell shape, and relative thickness vary among the four species in this study. The spongy mesophyll of the four species differ in number of cell layers. Because these variations can differentiate these species from one another, they are deemed to be useful in taxonomy. This study shows that anatomical features of the Medinilla species used in this study are related to their epiphytic nature and their occurrence in the forest understory. Keywords: Medinilla , epiphyte, sclerified mesophyll, hypodermis, druse, polocytic stomates

Anatomical characteristics and tissue localization of various flavonoid subclasses in Cotinus coggygria stem and leaf cross-sections

Leaves, twigs and wood of Cotinus coggygria (Anacardiaceae) are employed in European and Chinese traditional medicine to treat skin and mucosal injuries, fever, diarrhea, emesis and to enhance appetite. While being a popular medicinal plant, ancient tinctorial species and common ornamental shrub, its anatomical characteristics and tissue localization of major groups of active compounds have not been studied yet, to the best of our knowledge. Freehand cross sections of the stem at different developmental stages, leaf lamina and petiole were performed. Tissue types were studied using conventional dyes (toluidine blue; congo red and malachite green). Folin-Ciocalteu reagent, shift reagents (ammonia) and Naturstoffreagenz A were used to complement light microscopy and autofluorescence observations of flavonoid localization. Microscopes of type Optika XDS-3FL inverted fluorescence, and Olympus BH-2 for observation in visible light were employed. C. coggygria stem has a typical secondary structure. The bark contains resin ducts placed in the phloem; in younger twigs they are limited by outer sclerenchyma arches. Vascular cambium generates distinct annual rings, with a porous structure. The vessel frequency is of 100 – 200 mm2, and their perforations are simple. Medullar rays are narrow (1 – 2 cells). Chalcone and aurone derivatives are present in inner wood rings aged at least 2 years, in earlywood parts. Leaves display epidermis with anomocytic stomata on the lower surface. A hypodermis is present on the adaxial side. The midrib contains four main vascular bundles and cambium. The phloem encloses resin secretory cavities. The center of the midrib is occupied by parenchyma. Palisade parenchyma is two cells deep, and spongy mesophyll is situated towards the abaxial side. Reducing flavonoids are present in leaf mesophyll.

Laticifers identification and natural rubber characterization from the latex of Periploca angustifolia Labill. (Apocynaceae)

Periploca angustifolia (Apocynaceae) is a multipurpose shrub widely distributed in arid and semi-arid Tunisian regions. Local people often use this laticiferous shrub us a fodder for their livestock and consume the dried latex as a pleasant taste chewing. This study aims to identify laticifers distribution in vegetative and reproductive organs of Periploca angustifolia and investigate latex composition in order to valorize this constituent for later use. The histological study conducted on this species has confirmed the presence of laticifers in leaf (spongy and palisade mesophyll), stem (cortical parenchyma), root (periderm), floral bud (petal–carpel) and follicle (pericarp). Laticifers in the studied species are non-articulated. The purified rubber fraction from latex was subjected to different chemical analyses, proving that the rubber of P. angustifolia is composed of cis-1,4-polyisoprene as the main constituent. When the molecular weight distribution of rubber components in the exudate was measured using SEC analysis, the number-average molecular weight (Mn), the weight-average molecular weight (Mw), and polydispersity (Mw/Mn) were estimated to be Mn=2.3×105, Mw=4.1×105 and Mw/Mn=1.78, respectively. Thus, the obtained results show that the latex of P. angustifolia contains a natural rubber.