In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution^1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described^4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm¹, (2) a high survival rate of > 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days² and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter¹ in stable transgenic lines and the exon trap line FasII-GFP¹. (4) In contrast to other methods^4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days¹. The accompanying video details the function of individual parts of the in vivo imaging chamber^2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.     

A Complex Sub-cellular Component of Widespread Occurrence in Plant Latices

A survey of exudates from a number of latex-bearing plants revealsthe wide-spread occurrence of a complex subcellular componentapparently analogous to the ‘lutoid’ of Hevea brasiliensislatex. Under the conditions of our experiments this componentis usually associated with thread-like material.     

High Resolution Imaging of Plant Tissues10

Connelly, A., Lohman, J. A. B., Loughman, B. C., Quiquampoix,H. and Ratcliffe, R. G. 1987. High resolution imaging of planttissues by NMR.—J. exp. Bot. 38: 1713–1723. NMR images of living plant tissues were recorded at a ¹H frequencyof 200 MHz using a high resolution imaging technique that gavean in-plane pixel resolution of 50 µm x 50 µm orbetter. Images with interpretable contrast were obtained fromgerminating seeds, the roots of seedlings and the stems of youngplants. The expected structural features of these tissues werereadily observed including, in Mn²⁺ loaded tissue, the xylemvessels of maize root sections. Preliminary experiments on H₂O-D₂Oexchange in maize roots, on the uptake of Mn²⁺ by maize rootsand on the germination of seeds in situ demonstrate that thenon-invasive method of NMR mini-imaging has the potential tocomplement existing techniques for physiological investigationsin plant tissues. Key words: NMR imaging, water content, ion uptake     

植物果实的特异型启动子

植物果实特异型启动子能控制外源基因在果实中特异表达。目前已获得的果实特异型启动子主要包括E8、2A11/2A12、PG、MCPI、B33和ACC氧化酶启动子等。该文主要对这些果实特异性启动子及其存在的问题进行了总结,并对该领域今后的发展趋势进行了展望。     

植物抗病性物质的研究进展

植物与病原菌相互作用后在植物体内会产生抗性物质。植物的抗病性物质包括两大类 :植物固有的抗菌物质和植物保卫素。本文就近年来对植物抗病性物质的化学组成、生物合成、代谢途径方面的研究进行了综述。     

The Dry Preservation of Fixed Plant Material

A method for the dry-preservation of fixed plant material, root tips and buds, is described. The method seems to be advantageous on long expeditions and when material has to be sent away.

The material is transferred from the fixative to 70% alcohol (3 changes, 1/2 hour in the last). It is dried on blotting-paper. The dried material may be preserved a long time. Material kept dry for 4 1/2 years has proved to be quite satisfactory. Drying has been tried after fixation with CRAF-solutions (Webber and Randolph modifications) and fixatives containing osmic acid (Fleming-Benda and 2BD).

The dry material is swollen by keeping for 2 days in 10% alcohol. It is embedded in paraffin according to the usual method. A satisfactory staining has been obtained after these fixatives using iodine-gentian-violet and Feulgen stainings. In addition to chromosome counts dry material may be used for chromosome morphology studies.

Dried material fixed in aceto-alcohol (1:3) has not turned out to be specially suitable for squash preparations owing to the fragility of the chromosomes. If strong pressure is not applied, satisfactory results may, however, be obtained.     

Gas Chromatographic Determination of Phytol in Plant Material

Phytol can be gas chromatographed without decomposition on an all-glass column with 2% Hyprose SP-80 on Chromosorb W; is stationary phase. Utilizing this a technique has been developed by which phytol of plant origin can be quantitatively determined. The practical limit of sensitivity is about 0.02 μg of phytol.     

Freezing in the Preparation of Cleared Plant Material

In a study of chicory (Cichorium intybus) cross pollination it was necessary to investigate the penetration of germinated pollen tubes into stylar tissue.     

Visualizing Taste Papillae In Vivo with Scanning Electron Microscopy of a High Resolution Cast

A method using polyvinylsiloxane (PVS), a high-resolution dentalimpression material, to obtain negative images of lingual surfacesis described. Epoxy-resin tongue replicas made from these impressionswere examined with scanning electron microscopy (SEM). Thismethod has been developed to visualize structural details ofthe tongue surface of living human beings and laboratory animals.The utility of the method is demonstrated with hamster tongues,which have well-defined fungiform papillae with single tastepores, and human tongues, which have more variable surface structures.Replicas made from PVS impressions of tongues of living hamsterswere compared with the same tongues after fixation. The replicascontained much of the detail present in fixed tongues. WithSEM, it was possible to identify individual fungiform papillae,which contained depressions with the size and the location ofhamster taste pores. Individual papillae could also be recognizedin human-tongue replicas, but taste pores could not be identifiedwith certainty. These replicas provide permanent, three-dimensionalrecords of tongue topography that could be used to documentchanges due to trauma, disease and aging.     

Aceto-Iron-Haematoxylin for Staining Chromosomes in Squashes of Plant Material

Haematoxylin can be used successfully in the acetic squash technic if adequate mordanting is provided, (a) in the stain—composed of 4% haematoxylin and 1% iron alum in 45% acetic acid—and (b) in a step that combines additional fixation, mordanting and maceration in a 1:1 HCl-alcohol mixture, to which is added chrome alum, iron alum and iodic acid: 0.1 gm of each to 6 ml of HCl-alcohol. The material is usually given a preliminary fixation in 1:3 acetic alcohol, then macerated, fixed and mordanted in the acidified alum-HIO₃ step for 10 min, transferred to Carney's fluid (6:3:1) for 10-20 min, squashed in a drop of stain and gently heated. In some species, the preliminary fixation may be omitted. The method yields intensely and selectively stained chromatin. To secure consistently good results, the stain can be diluted with 45% acetic acid, and the iodic acid omitted for some plant materials.     

Inhibition of Fungal and Bacterial Plant Pathogens In Vitro and In Planta with Ultrashort Cationic Lipopeptides

Plant diseases constitute an emerging threat to global food security. Many of the currently available antimicrobial agents for agriculture are highly toxic and nonbiodegradable and cause extended environmental pollution. Moreover, an increasing number of phytopathogens develop resistance to them. Recently, we have reported on a new family of ultrashort antimicrobial lipopeptides which are composed of only four amino acids linked to fatty acids (A. Makovitzki, D. Avrahami, and Y. Shai, Proc. Natl. Acad. Sci. USA 103:15997-16002, 2006). Here, we investigated the activities in vitro and in planta and the modes of action of these short lipopeptides against plant-pathogenic bacteria and fungi. They act rapidly, at low micromolar concentrations, on the membranes of the microorganisms via a lytic mechanism. In vitro microscopic analysis revealed wide-scale damage to the microorganism's membrane, in addition to inhibition of pathogen growth. In planta potent antifungal activity was demonstrated on cucumber fruits and leaves infected with the pathogen Botrytis cinerea as well as on corn leaves infected with Cochliobolus heterostrophus. Similarly, treatment with the lipopeptides of Arabidopsis leaves infected with the bacterial leaf pathogen Pseudomonas syringae efficiently and rapidly reduced the number of bacteria. Importantly, in contrast to what occurred with many native lipopeptides, no toxicity was observed on the plant tissues. These data suggest that the ultrashort lipopeptides could serve as native-like antimicrobial agents economically feasible for use in plant protection.