Lateral Root Inducible System in Arabidopsis and Maize

Summary

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Abstract

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Introduction

The root system is crucial for plant growth, since it ensures anchorage and uptake of water and nutrients from the soil. Because the expansion of a root system mainly relies on the production of lateral roots, their initiation and formation have been widely studied. Lateral roots are initiated in a specific subset of pericycle cells, called founder cells¹. In most dicots, such as Arabidopsis thaliana, these cells are located at the protoxylem poles², whereas in monocots, such as maize, they are found at the phloem poles³. Founder cells are marked by an increased auxin response⁴, followed by expression of specific ce....

Protocol

1. Arabidopsis LRIS Protocol

Note: The text refers to "small" or "large" scale experiments. Small scale experiments, such as marker line analysis and histological staining^{6, 14}, require only a few samples. Large scale experiments, such as quantitative real-time qRT-PCR, micro-arrays^9-11 or RNA sequencing, require a larger amount of samples. As such, an amount of ~1000 seedlings per sample was used by Vanneste et al.¹¹ to perform microarray experiment after root segment dissection.

DAY 1

1. Sterilization of Ara....

Results

Application of the LRIS to Perform Comparative Transcriptomics of the Lateral Root Initiation Process

One application of the LRIS is the comparison and correlation of gene expression profiles during lateral root formation in different species. Comparative transcriptomics approaches create the possibility to pinpoint orthologous genes involved in the lateral root development process in different species. Lateral .......

Discussion

In the Arabidopsis LRIS protocol, it is important to only transfer the seedlings that have grown entirely in contact with the NPA-containing growth medium. This ensures that lateral root initiation is blocked over the entire root length. In order to prevent wounding the plantlets during transfer, the arms of the curved forceps can be hooked under the cotyledons of the seedling. Upon transfer, make sure that the seedling roots are in sufficient contact with the NAA-containing agar medium. This can be achieved by .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ARABIDOPSIS LRIS
Seeds
Arabidopsis seeds			Col-0 ecotype
Gas sterilization of seeds
micro-centrifuge tubes 1.5 ml	SIGMA-ALDRICH	0030 125.215	Eppendorf microtubes 3810X, PCR clean
micro-centrifuge tubes 2 ml	SIGMA-ALDRICH	0030 120.094	Eppendorf Safe-Lock microcentrifuge tubes
hydrochloric acid	Merck KGaA	1,003,171,000	37% (fuming) for analysis EMSURE ACS,ISO,Reag. Ph Eu
glass desiccator	SIGMA-ALDRICH		Pyrex
glass beaker
plastic micro-centrifuge tubes box or holder
Bleach sterilization of seeds
ethanol	Chem-Lab nv	CL00.0505.1000	Ethanol, abs. 100% a.r. dilute to 70%
sodium hypochlorite (NaOCl)	Carl Roth	9062.3	12%
Tween 20	SIGMA-ALDRICH	P1379
sterile water
Growth medium
Murashige and Skoog salt mixture	DUCHEFA Biochemie B.V.	M0221-0050
myo-inositol	SIGMA-ALDRICH	I5125-100G
2-(N-morpholino)ethanesulfonic acid (MES)	DUCHEFA Biochemie B.V.	M1503.0100
sucrose	VWR, Internation LLC	27483.294	D(+)-Sucrose Ph. Eur.
KOH	Merck KGaA	1050211000	pellets for analysis (max. 0.002% Na) EMSURE ACS,ISO,Reag. Ph Eur
Plant Tissue Culture Agar	LabM Limited	MC029
Lateral root induction chemicals
N-1-naphthylphthalamic acid (NPA)	DUCHEFA Biochemie B.V.	No. N0926.0250	10 µM (Arabidopsis)
1-naphthalene acetic acid (NAA)	DUCHEFA Biochemie B.V.	No. N0903.0050	10 µM (Arabidopsis)
dimethylsulfoxide (DMSO)	SIGMA-ALDRICH	494429-1L
Making a mesh for transfer
nylon mesh	Prosep byba		Synthetic nylon mesh 20 µm
Sowing and seedling handling
square petri dish plates	GOSSELIN	BP124-05	12 x 12 cm
50 ml DURAN tubes	SIGMA-ALDRICH	CLS430304	Corning 50 ml centrifuge tubes
drigalski	Carl Roth	K732.1
pipette
cut pipette tips	Daslab	162001X	Universal 200, cut off 5 mm of tip before autoclaving
breathable tape	3M Deutschland GmbH	cat. no. 1530-1
tweezers	Fiers nv/sa	K342.1; K344.1	Dumont tweezers type a nr 5; Dumont tweezers type e nr 7
Growth conditions
growth room			21 °C, continuous light
Materials	Company	Catalog	Comments
MAIZE LRIS
Seeds
Maize kernels			B-73
Bleach sterilization of kernels
glass beaker
magnetic stirrer	Fiers nv/sa	C267.1
sodium hypochlorite (NaOCl)	Carl Roth	9062.3	12%
sterile water
Lateral root induction chemicals
N-1-naphthylphthalamic acid (NPA)	DUCHEFA Biochemie B.V.	No. N0926.0250	50 µM (maize primary root), 25 µM (maize adventitious root)
1-naphthalene acetic acid (NAA)	DUCHEFA Biochemie B.V.	No. N0903.0050	50 µM (maize)
dimethylsulfoxide (DMSO)	SIGMA-ALDRICH	494429-1L
Sowing and seedling handling
paper hand towels	Kimberly-Clark Professional*	6681	SCOTT Hand Towels - Roll / White; sheet size (24 x 46 cm)
seed germination paper	Anchor Paper Company		10 X 15 38# seed germination paper
tweezers	Fiers nv/sa	K342.1; K344.1	Dumont tweezers type a nr 5; Dumont tweezers type e nr 7
250 ml (centrifuge) tubes	SCHOTT DURAN	2160136	approx. 5.6 cm diameter and 14.7 cm height
700 ml tubes	DURAN GROUP	213994609	cylinders, round foot tube, D 60  x 250
rack			for maize tubes, home made
sterile water
Growth conditions
growth cabinet			27 °C, continuous light, 70% relative humidity