A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Summary

Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants.

Abstract

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of electrical transmission between distant cells in an organism requires in vivo, artifact-free recordings of cells embedded within their native environment. The transmission of electrical signals from wounded to unwounded areas in a plant has since long piqued the interest of botanists. The phloem, the living part of the plant vasculature that is spread throughout the plant, has been postulated as a major tissue in electrical transmission in plants. The lack of suitable electrophysiological methods poses many challenges for the study of the electrical properties of the phloem cells in vivo. Here we present a novel approach for intracellular electrophysiology of sieve elements (SEs) that uses living aphids, or other phloem-feeding hemipteran insects, integrated in the electrical penetration graph (EPG) circuit. The versatility, robustness, and accuracy of this method made it possible to record and study in detail the wound-induced electrical signals in SEs of central veins of the model plant Arabidopsis thaliana¹. Here we show that EPG-electrodes can be easily implemented for intracellular electrophysiological recordings of SEs in marginal veins, as well as to study the capacity of SEs to respond with electrical signals to several external stimuli. The EPG approach applied to intracellular electrophysiology of SEs can be implemented to a wide variety of plant species, in a large number of plant/insect combinations, and for many research aims.

Introduction

The ability to produce long-distance electrical signals is an advantageous trait of multi-cellular organisms that allows for efficient responses to external stimuli. This trait has evolved independently in plants and animals, and thus represents a case of convergent evolution. Given that electrical signals are coupled with important functions in animals such as neural transmission and muscle contraction, the molecular basis, mechanism of transmission, and function of stimulus-induced electrical signals in animals are subjects of intensive research. In contrast, stimulus-induced electrical signaling in plants has received little research attention. Although plants have....

Protocol

1. Aphid Rearing

Note: The choice of plant and aphid species for EPG recordings depends on the research aim. For studies on Arabidopsis thaliana, the aphid Brevicoryne brassicae is appropriate.

1. Rear B. brassicae aphids in a greenhouse on Brassica oleracea. Keep the plants used for aphid rearing in cages, in order to avoid contaminating other plants. Keep aphid-rearing plants and experimental plants (in our case B. oleracea and A. thaliana) in separate rooms, in order to avoid contamination of experimental plants with aphids.
2. Transfer aphids to fresh plants about eve....

Results

In a previous study, we implemented the EPG-electrode technique with the purpose of characterizing the electrical signals produced in SEs of the midvein during caterpillar attack¹. The midvein is a preferred insertion site for conventional glass electrodes, as well as for glass-stylet electrodes, because it is SE-dense, and relatively robust, hence amenable to the fixation needed for implementing these techniques. Here, we took advantage of the versatility of the EPG electrode with the purpose of gathering ele.......

Discussion

This article provides a detailed protocol for making Electrical Penetration Graph (EPG) recordings. The EPG technique is well established, with 100-200 active users worldwide, and it has been implemented for many studies on different topics, for example: a) host plant resistance to aphids and other stylet-bearing insects¹³; b) plant virus and pathogen transmission mechanisms¹⁴; c) insecticide mode of action, (toxicity and behavior changes)¹⁵; d) EPGs have even been useful to demonstrate t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Brass connector pins	EPG Systems/hardw.shop		Φ 1.2 mm
Thin copper wire	EPG Systems/hardw.shop		approx. Φ 0.2 mm
Thin gold wire	EPG Systems		Φ 18 µm
Soldering fluid	hardware shop		matching the soldering wire
Resin-cored soldering wire	hardware shop
Styrofoam	any
Water-based silver glue	EPG Systems		recipe in: www.epgsystems.eu
Paper wipes	Kimberly-Clark	5511
Soldering bolt	any
Stereomicroscope	Hund Wetzlar		minimum magnification is 10X
Small scissors	Fine Science Tools	14088-10
Scalpel	Fine Science Tools	10050-00
Fine forceps	Fine Science Tools	11231-20
Vortex	A. Hartenstein	L46
Watercolor brushes	any		Number 1 or 2
Air suction device			see description in: www.epgsystems.eu
Insect pins	any		No. 1 or 2
Solid table
Faraday cage			Hand made
Computer	Fujitsu Siemens
Data acquisition software	EPG Systems		Stylet+d
Giga-4 (-8) Complete System	EPG Systems
includes the following:
Main control box with USB output		Di155/Di710	12/14 bit, rate 100 Hz (softw. fixed)
EPG probes 4 (8)			50x DC pre-amplifier
Swivel clamps on rod
DC power adaptor			bipolar, 230/115 VAC to -/+8 VDC
Plant electrodes and cables
Additional test and ground cables