Phloem Methods and Protocols

This detailed book presents methods that enable the determination of parameters relevant to phloem research in the most efficient ways, putting plant scientists with access to adequate instrumentation in the position to answer any phloem-related question. The collection explores techniques that have been used for decades, such as tracing phloem transport with carbon isotopes, as well as recent developments, such as esculin-based assays of phloem transport and super-resolution microscopy of phloem proteins. As such, the book presents the state-of-the-art in phloem research and, at the same time, a starting point for the development of new methods that will fill the remaining gaps in the phloem researcher’s toolbox in the future. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and tips on troubleshooting and avoiding known pitfalls.

 Authoritative and comprehensive, Phloem: Methods and Protocols is an ideal guide for all researchers exploring this challenging area of plant biology.

Part 1 : Investigating phloem anatomy.- Optimal preparation of tissue sections for light-microscopic analysis of phloem anatomy.- Transmission electron microscopy of the phloem with minimal artifacts.- Scanning electron microscopy of the phloem.- Non-invasive investigation of phloem structure by 3D synchrotron X-ray microtomography.- Quantification of leaf phloem anatomical features with microscopy.- Methods of phloem visualization - a clear future in sight?.- Part 2 : Visualizing phloem proteins.- Super-resolution microscopy of phloem proteins.- Live-cell imaging of fluorescently-tagged phloem proteins with confocal microscopy.- Immunohistochemical localization of proteins in the phloem: problems and solutions.- Part 3 : Sampling phloem sap.- SStudying phloem loading with EDTA-facilitated phloem exudate collection and analysis.- Measurement of inner bark and leaf osmolality.- Using 13C to quantify phloem transport on tall plants in the field .- Non-invasive determination of phloem transport speed with carbon-14 (14C).- Detecting rapid changes in carbon transport and partitioning with  carbon-11 (11C).- In vivo veritas:  radiotracers in studies of phloem transport of carbohydrate.- Measuring phloem transport velocity in Arabidopsis seedlings using the 

fluorescent coumarin glucoside, esculin.- Measuring phloem transport velocity on a tissue-level using a phloem-mobile dye.- Part 4 : Investigating mechanism and quantities of phloem loading and unloading.- Quantification of symplasmic phloem loading capacity with live-cell microscopy.- Assessing Long-distance Carbon Partitioning from Photosynthetic Source Leaves to Heterotrophic Sink Organs with Photoassismilated [14C]CO2.- Measurement of subcellular metabolite concentrations in relation to phloem loading.- Measuring sucrose transporter activity using a protoplast esculin assay.- The AtSUC2 promoter: A powerful tool to study phloem physiology and development.- Part 5 : Phloem physiology.- Using aphids to measure turgor pressure inside sieve elements.- Stem compression: A means to reversibly reduce phloem transport in tree stems.- Methods for assessing the role of phloem transport in plant stress responses.- Part 6 : Theoretical models and microfluidics of phloem function.- Modeling the hydraulic conductivity of phloem sieve elements.- Using a multi-compartmental metabolic model to predict carbon allocation in Arabidopsis thaliana.- A mechanistic model to predict distribution of carbon among multiple sinks.- Using a mathematical model of phloem transport to optimize strategies for crop improvement.- Making microfluidic devices that simulate phloem transport.- Part 7 : Signals in the phloem.- Efficient establishment of interfamily heterograft of Nicotiana benthamiana and Arabidopsis thaliana.- Identification of phloem mobile mRNAs using the Solanacea heterograft system.- Analyzing and predicting phloem mobility of macromolecules with an online database.- Measurement of electropotential waves in intact phloem sieve elements using microelectrodes.- Measurement of electropotential waves in intact sieve elements using aphids as bioelectrodes.- What can cell culture systems reveal about sieve element differentiation?