Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

Summary

The goal of this protocol is to show how to load the CFDA into different sites of the bottom parts of Arabidopsis. We then present the resulting distribution pattern of CF in the shoots.

Abstract

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, phloem transport and vascular patterning. This protocol shows bottom-to-top carboxyfluorescein (CF) movement in the Arabidopsis by using the root-cutting and the hypocotyl-pinching procedure respectively. These two different procedures result in different efficiencies of CF movement: about 91% appearance of CF in the shoots with the hypocotyl-pinching procedure, whereas only about 70% appearance of CF with the root-cutting procedure. The simple change of loading sites, resulting in significant changes in the mobile efficiency of this symplastic dye, suggests CF movement might be subject to the symplastic regulation, most probably by the root-hypocotyl junction.

Introduction

Many fluorescent tracers with a range of spectral properties, such as 5(6)-carboxyfluorescein (CF)¹, 8-hydroxypyrene-1,3,6-trisulphonic acid², Lucifer yellow CH (LYCH)³, Esculin and CTER⁴, have been developed and applied in plants to monitor symplastic movement and phloem activity. Generally, a symplastic dye is loaded into a cut in the target tissue and the sequential dispersion of the reporter into other parts of plant will demonstrate the intercellular communication. Although the mechanism of dye absorption is not fully understood, the principle underlying CF movement in....

Protocol

1. Arabidopsis vertical growth in MS medium

1. The interior of laminar flow cabinet needs to be treated with 30 min UV light and 15 min airflow before usage. Make sure to close the glass door when UV light is on. Spray all tools and plates with 70% ethanol before placing them in the cabinet.
2. Prepare the Murashige and Skoog (MS) medium in a standard 90 mm-diameter Petri dish under a laminar flow cabinet.
   NOTE: The MS medium contains the following components: 20.6 mM NH₄NO₃, 18.8 mM KNO₃, 1.25 mM KH₂PO₄, 1.5 mM MgSO₄·7H₂O, 3 mM CaCl₂·2H

Results

Symplastic movement is often subject to environmental fluctuations. Perturbation of the plant growing state, and even the process of tissue preparation will affect the size exclusion limit of plasmodesmata, thus affecting the symplastic transport²². To improve the staining efficiency, we confine our operation in the growth room, where the temperature and moisture is tightly controlled, and also perform the whole procedure as quickly as possible (ideally within 10-1.......

Discussion

Emerging studies have shown that plants can rapidly respond to external stimuli²³, including manipulation introduced to the experimental procedures²². In our initial experiment, our oversight of this knowledge often leads to staining failure. With these lessons, we suggest that the following precautions should be kept in mind when performing this experiment: (1) the seeds after harvest should be kept in a storage cabinet set to a low temperature and low moisture; (2) manipu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
KNO3	Sinopharm Chemical Reagent	10017218
KH2PO4	Sinopharm Chemical Reagent	10017608
MgSO4·7H2O	Sinopharm Chemical Reagent	10013018
CaCl2·2H2O	Sinopharm Chemical Reagent	20011160
MnSO4·H2O	Sinopharm Chemical Reagent	10013418
Na2MoO4·2H2O	Sinopharm Chemical Reagent	10019818
Boric Acid	Sinopharm Chemical Reagent	10004818
ZnSO4·7H2O	Sinopharm Chemical Reagent	10024018
CuSO4·5H2O	Sinopharm Chemical Reagent	10008218
CoCl2·6H2O	Sinopharm Chemical Reagent	10007216
KI	Sinopharm Chemical Reagent	10017160
FeSO4·7H2O	Sinopharm Chemical Reagent	10012118
EDTA	Sinopharm Chemical Reagent	10009717
NaOH	Sinopharm Chemical Reagent	10019718
KOH	Sinopharm Chemical Reagent	10017018
Sucrose	Sinopharm Chemical Reagent	10021418
Myo-inositol	MACKLIN	I811835
Nicotinic Acid	MACKLIN	N814565
Pyridoxine HCl	MACKLIN	V820447
Thiamine HCl	MACKLIN	T818865
Glycine	MACKLIN	G800880
Agar powder	Novon	ZZ14022
Fluorescence Microscope	Zeiss	Axio Zoom V16
Dissecting microscope	SDPTOP	SRE-1030
200μl pipette	Dragon Laboratory Instruments	713111110000-20-200ul
2.5μl pipette	Eppendorf	3120000011
Fine forceps	TWEEZERS	ST-15
Parafilm	PARAFILM	PM-996
Stainless steel double-sided blade	Gillette	Platinum-Plus Double-Edge Blade