Adjacent Leaf Tissues Research Articles

Application of fluorescence microscopy for histological discrimination of golden camellias leaves

Histological analysis, which aims to investigate the microscopic anatomy of biological tissues, has been a simple and powerful technique for plant taxonomy. Sectioning followed by staining methods is widely used in observing histological structures. However, the staining techniques often destroy tissue and provide low-quality images due to nonspecific reactions with the dyes making further analysis difficult. In this report, we propose an applicable non-staining histology protocol based on auto-fluorescence characteristics of plant tissues and its application in the anatomical discrimination of six similar-appearance species of golden camellias as a case study. We compared the images from the same tissue under a bright field with the staining step and under fluorescence directly without the staining step in the sample preparation. The images were taken from Eclipse Ni-U microscopy (Nikon, Japan) with a color DS-Ri2 camera (Nikon, Japan) and NIS-ELEMENTS Basic Research Imaging software. The non-staining method demonstrated significant advantages compared to the staining protocol. The fluorescent images showed the distinction between adjacent leaf tissues with their own naturally reflective colors. In addition, the anatomical parameters, including the xylem area, phloem area, bundle sheath area, and palisade/spongy width ratio, were easily measured in good-quality images. These parameters were used in discriminative analysis by the Principal Component Analysis (PCA). The PCA diagram demonstrated the separation of six species, thus suggesting that these anatomical parameters can be used for taxonomy. In conclusion, our study showed a helpful technique in histological analysis that significantly contributes to the taxonomy of golden camellias species and can be applied in other plant varieties.

Spatiotemporal heterogeneity of tomato induced defense responses affects spider mite performance and behavior

ABSTRACTWhen feeding from tomato (Solanum lycopersicum), the generalist spider mite Tetranychus urticae induces jasmonate (JA)- and salicylate (SA)-regulated defense responses that hamper its performance. The related T. evansi, a Solanaceae-specialist, suppresses these defenses, thereby upholding a high performance. On a shared leaf, T. urticae can be facilitated by T. evansi, likely via suppression of defenses by the latter. Yet, when infesting the same plant, T. evansi outcompetes T. urticae. Recently, we found that T. evansi intensifies suppression of defenses locally, i.e., at its feeding site, after T. urticae mites were introduced onto adjacent leaf tissue. This hyper-suppression is paralleled by an increased oviposition rate of T. evansi, probably promoting its competitive population growth. Here we present additional data that not only provide insight into the spatiotemporal dynamics of defense induction and suppression by mites, but that also suggest T. evansi to manipulate more than JA and SA defenses alone.

Differentially Expressed Genes in Hypericin-Containing Hypericum perforatum Leaf Tissues as Revealed by De Novo Assembly of RNA-Seq

Hypericum perforatum is a traditional medicinal plant used for various purposes since ancient times because of the valuable secondary metabolites. The genomic information for H. perforatum is limited and the regulatory networks of secondary metabolism are still unknown. The naphthodianthrone hypericin is the metabolite of our main interest. It is a red-colored photodynamic pigment localized in dark glands of some Hypericum species. High-throughput sequencing technology, especially RNA-Seq, followed by de novo assembly and analysis of differential gene expression provides an important tool for functional genomics of non-model organisms. It represents an opportunity of insight into dynamic biological processes including those of secondary metabolism. Transcriptome analysis followed by the analysis of differential gene expression of H. perforatum leaf tissues containing and lacking dark glands was performed to identify the genes involved in hypericin biosynthesis and to profile expression patterns in greater detail. A total of 18.53 G of cleaned read nucleotides were generated and assembled into 139,959 contigs with N50 of 1801 bp. Among them, 66,817 (47.74 %) contigs were annotated. Differentially expressed genes were discovered by comparison of dark glands with adjacent leaf tissue and contrasting inner part leaf tissue without dark glands. A total of 799 upregulated genes were found in the tissues containing dark glands and 263 enzymes were identified, including candidate genes of hypericin biosynthesis, especially the genes coding for polyketide synthases and those involved in defense reactions. This study determined candidate genes involved in hypericin biosynthesis providing a valuable source for perspective metabolic engineering of bioactive substances.

Transgene Silencing and Transgene-Derived siRNA Production in Tobacco Plants Homozygous for an Introduced AtMYB90 Construct

Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2–R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3′ half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production.

A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies

BackgroundSome plants inhabiting saline environment remove salts via the salt glands embedded in the epidermal tissues. Cytological studies of salt glands will provide valuable information to our understanding of the secretory process. Previous studies on salt gland histology relied mainly on two-dimensional microscopic observations of microtome sections. Optical sectioning properties of confocal laser scanning microscope offer alternative approach for obtaining three-dimensional structural information of salt glands. Difficulty in light penetration through intact leaves and interference from neighbouring leaf cells, however, impede the acquiring of good optical salt gland sections and limit its applications in salt gland imaging. Freeing the glands from adjacent leaf tissues will allow better manipulations for three-dimensional imaging through confocal laser scanning microscopy.ResultsHere, we present a simple and fast method for the isolation of individual salt glands released from the interference of neighbouring cells. About 100-200 salt glands could be isolated from just one cm2 of Avicennia officinalis leaf within hours and microscopic visualization of isolated salt glands was made possible within a day. Using these isolated glands, confocal laser scanning microscopic techniques could be applied and better resolution salt gland images could be achieved. By making use of their intrinsic fluorescent properties, optical sections of the gland cells could be acquired without the use of fluorescent probes and the corresponding three-dimensional images constructed. Useful cytological information of the salt gland cells could also be obtained through the applications of fluorescent dyes (e.g., LysoTracker® Red, FM®4-64, Texas Red®).ConclusionsThe study of salt glands directly at the glandular level are made possible with the successful isolation of these specialized structures. Preparation of materials for subsequent microscopic observations of salt glands could be achieved within a day. Potential applications of confocal fluorescence microscopic techniques could also be performed using these isolated glands. Experiments designed and targeted directly at the salt glands were explored and cytological information obtained herein could be further incorporated towards the understanding of the mechanism underlying secretion in plant salt glands.

Heating the ends of leaves cut during coring of whole heads of lettuce reduces subsequent phenolic accumulation and tissue browning

Removal of the central axis or core of head lettuce harvested for the fresh-cut market (i.e., coring) is done in the field to reduce transit weight and wastage at the processing plant. Wounding done to the base of the leaves during coring induces an increase in phenolic metabolism and subsequent browning of the injured and adjacent leaf tissue. Applying a moist heated surface (e.g., 55 °C) to the tissue freshly exposed by coring for 10–15 s significantly reduced phenolic accumulation and subsequent browning. This inhibition persisted for 6 days at 10 °C and was not accompanied by increased decay of the treated tissue. Wetting the heated surface with an inhibitor of wound-induced phenolic metabolism (e.g., C6 or C8 mono-carboxylates) reduced the accumulation of wound-induced phenolic compounds at marginal or non-heat-shock inducing temperatures, but not at heat-shock inducing temperatures that significantly reduced the wound response by themselves. Heat-shocking the freshly exposed tissue during coring may be an effective method to reduce subsequent browning of the injured and adjacent lettuce leaf tissue.

Enzyme-Linked Immunosorbent Assay Testing of Shoots Grown In Vitro and the Use of Immunocapture-Reverse Transcription-Polymerase Chain Reaction Improve the Detection of Prunus necrotic ringspot virus in Rose

We developed and evaluated two different methods to improve the detection of the most prevalent virus of rose in Europe, Prunus necrotic ring-spot virus (PNRSV). Immunocapture-reverse transcription-polymerase chain reaction was estimated to be about 100 times more sensitive than double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and showed an equivalent specificity. Based on the observation that PNRSV multiplies actively in young growing tissues (axillary shoots and cuttings), an in vitro culture method allowing rapid (about 15 days) and homogeneous development of dormant axillary buds with high virus titers was standardized. ELISA tests of these young shoots showed, in some cases, a 10(4) to 10(5) increase in sensitivity in comparison to adjacent leaf tissues from the rose mother plants. Between 21 and 98% (depending on the season) more samples were identified as positive by using ELISA on samples from shoot tips grown in vitro rather than on leaves collected directly from the PNRSV-infected mother plants. This simple method of growing shoot tips in vitro improved the confidence in the detection of PNRSV and eliminated problems in sampling appropriate tissues.

Apoplastic and symplastic pathways in leaves and roots of the seagrass Halodule uninervis (Forssk.) Aschers.

The apoplastic tracers Cellufluor and lanthanum nitrate, and the symplastic tracer uranin, were used to study pathways for transport in leaves and roots of the seagrass Halodule uninervis (Forssk.) Aschers. An apoplastic pathway was shown to exist in most leaf tissues except in the region of the median vein where Casparian bands in bundle sheath cell walls prevented the penetration of the tracers into vascular tissues. Entry of uranin into leaf tissues was limited. The tracer remained in the protoplast of leaf epidermal cells and did not move symplastically into adjacent leaf tissues. Casparian bands in walls of root exodermal cells blocked further apoplastic movement of tracers into root tissues. In contrast, a symplastic pathway was shown to exist from the water column to the root cortex. The results are discussed in relation to the significance of apoplastic and symplastic transport in this seagrass.

Physiological Effects of Galls Induced by Phylloxera notabilis (Homoptera: Phylloxeridae) on Pecan Foliage

Effect of the Phylloxera notabilis Pergande gall was assessed on leaf chlorophyll, leaf nutrient status, and leaf gas exchange of nursery pecan, Carya illinoensis (Wangenh.) C. Koch, ‘Stuart’. Gall tissue was significantly different from ungalled tissue in every nutrient investigated. Galls contained 60% N, 50% Ca, and 50% Al, but 260% K, 150% P, and 200% Na of leaf tissue from uninfested (control) leaflets. Analysis of concentric rings of tissue revealed that chlorophyll and all macronutrients except P were altered in leaf tissue 0–1.5 cm away from a gall compared with control leaflets. Net CO2 assimilation rate of infested tissue was reduced by an average of 27%, which was equivalent to almost 4-fold the percentage of gall area occurring in the field of measurement. Significant reductions in leaf conductance and transpiration also occurred over leaf tissue containing a gall. Cumulative gall cross-sectional area was negatively correlated with net CO2 assimilation rate. Thus, not only did the P. notabilis gall divert resources away from normal metabolic function, but it also interfered with the nutrient status and photosynthetic capacity of adjacent leaf tissue.

Evaporation from rain drops on leaves in a cereal canopy: A simulation model

In crop canopies, the persistence of discrete water drops on leaves after rain is of particular importance to the epidemiology of certain foliar pathogens. A model is described which simulates the heat and water vapour fluxes in a plant canopy and includes evaporation from water drops on the leaves. Energy balance equations allow for heat conducted to drops from the adjacent leaf tissue. Preliminary field tests of model performance for winter wheat, which compare predicted and visually assessed leaf wetness, are encouraging.

Olive leaf spot and its control with fungicides

In Brief Olive leaf spot, caused by the fungus Cycloconium oleaginum Cast., has recently been prevalent in California. The first symptoms are inconspicuous sooty blotches on the leaf blade. As these enlarge, the rest of the leaf turns yellow. Severe defoliation follows leaf infection. In some orchards 10 to 20 per cent of the fruiting twigs died as a result of defoliation. The fungus was isolated by transferring single ungerminated conidia to a solid medium. It proved to be extremely slow-growing. Only three of many isolates produced conidia in culture. Conidia germinated and mycelium grew between 9° and 30° C, the optimum being 16° to 20°. New lesions developed in the fall one season in central California, but not until late January and February in three other seasons. Affected leaves became yellow and fell during May and early June. Lesions on leaves that remained in the tree seem to be the chief if not the only source of inoculum. These bore numerous conidia in spring but few during the summer. In autumn the fungus grew from the margins of such lesions into adjacent leaf tissue and produced conidia; thus a supply of inoculum was insured at a time when moisture conditions were favorable to infection. The spores seem to be spread by rain more than by air currents. Bordeaux mixture (8-8-100 or 10-10-100) applied in fall or early winter effectively prevented leaf infection in three out of four seasons. Later applications gave less satisfactory control. For two seasons one treatment of 8-8-100 bordeaux proved less efficacious than two treatments. In the next two seasons, however, one November treatment of 10-10-100 bordeaux gave adequate protection. On the whole, proper timing of the bordeaux treatment seemed more important than renewing the fungicide deposit. Ferric dimethyl dithiocarbamate and zinc dimethyl dithiocarbamate sprays containing an oil supplement (“sticker”) reduced infection materially but not so effectively as bordeaux. Yellow cuprous oxide in a two-treatment program reduced infection satisfactorily but injured the leaves. Lime-sulfur (3 per cent) controlled leaf spot as effectively as 10-10-100 bordeaux. Persistent effects of treatments were sometimes noted. In several orchards trees sprayed with bordeaux or lime-sulfur in only one season bore less leaf spot at the end of the next season than did unsprayed trees.