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 cell cycle genes (e.g., CYCLIN B1;1 / CYCB1;1), after which they undergo a first round of asymmetric anticlinal divisions⁵. After a series of coordinated anticlinal and periclinal divisions, a lateral root primordium is formed that finally will emerge as an autonomous lateral root. The location and timing of lateral root initiation are however not predictable, since these events are neither abundant nor synchronized. This impedes the use of molecular approaches such as transcriptomics to study this process.

To tackle this, a Lateral Root Inducible System (LRIS) has been developed^{6, 7}. In this system, seedlings are first treated with N-1-naphthylphthalamic acid (NPA), which inhibits auxin transport and accumulation, consequently blocking lateral root initiation⁸. By subsequently transferring the seedling to medium containing the synthetic auxin 1-naphthalene acetic acid (NAA), the entire pericycle layer responds to the elevated auxin levels thereby massively inducing lateral root initiating cell divisions⁶. As such, this system leads to fast, synchronous and extensive lateral roots initiations, allowing easy collection of root samples enriched for a specific stage of lateral root development. Subsequently, these samples can be used to determine genome-wide expression profiles during lateral root formation. The LRIS has yielded already significant knowledge about lateral root initiation in Arabidopsis and maize^9-13, but the need to apply this system to other plant species becomes more apparent as more genomes are sequenced and there is an increasing interest to transfer knowledge to economical important species.

Here, the detailed protocols of the Arabidopsis and maize LRISs are given. Next, an example of the use of the system is provided, by illustrating how transcriptomics data gained from the maize LRIS can be used to identify functional homologs that have a conserved function during lateral root initiation across different plant species. Finally, guidelines to optimize the LRIS for other plant species are proposed.

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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 Arabidopsis Seeds
   Note: Select one of the following procedures for seed sterilization. Any of them is suitable for the next steps of the protocol. Gas sterilization (1.1.2) presents two main advantages over liquid sterilization (1.1.1): it takes less time when handling a large number of seeds, since no pipetting has to occur for the individual samples; and the sterilized seeds can be stored for a longer period. However, it requires a desiccator and careful handling.
   1. Liquid sterilization
      1. Pour the desired number of seeds in micro-centrifuge tubes. Use up to 20 mg (~800 seeds) in a 1.5 ml tube or 40 mg (~1600 seeds) in a 2 ml micro-centrifuge tubes.
      2. Add 1 ml of 70% ethanol. Invert or gently shake for 2 min.
      3. Replace the 70% ethanol with 1 ml sterilization solution for 15 min. (Prepare fresh sterilization solution: 3.85 ml sodium hypochlorite (NaOCl) stock (12%) (CAUTION: Use fume hood), 5 µl Tween 20, up to 10 ml with water. Invert the tubes several times to make sure that all seeds come in contact with the solution.
      4. In a laminar air flow cabinet, replace the sterilization solution with 1 ml sterile distilled water and rinse the seeds 5 times for 5 min by repeatedly replacing with 1 ml of fresh sterile distilled water.
   2. Gas Sterilization
      1. Pour the desired number of seeds in a 2 ml micro-centrifuge tube. Use individual tubes when working with several independent lines, and arrange them opened in a plastic micro-centrifuge tubes box or holder. If the number of seeds in one tube exceeds 500 (12.5 mg), subdivide the seeds in the appropriate number of tubes to ensure successful sterilization. Make sure the caps of neighboring tubes do not cover each other. Fit together the lid of the box on the bottom, as it needs to be in the desiccator as well for sterilization.
      2. Place the box in the bowl of a desiccator, along with a glass beaker (CAUTION: Use fume hood).
      3. Fill the beaker with 100 ml of 12% NaOCl (CAUTION: Use fume hood). Put the lid on the desiccator, but leave a small aperture where the beaker is placed.
      4. Add 3 ml of 37% hydrochloric acid (fuming) (CAUTION: Use fume hood) to the beaker through the small aperture and quickly close the desiccator. The solution will bubble for some time due to Cl₂-gas formation. Leave the system closed O/N (or at least 8 hr).
      5. Remove the lid of the desiccator. Take out the box and cover it with the lid to avoid contamination during transport.
      6. Put the box in a laminar air flow cabinet, remove the lid and leave the seeds for about 1 hr at RT to allow gas release.
         Note: Seeds can be safely stored dry in closed micro-centrifuge tubes for up to 2 weeks at 4 °C.   
2. Lateral Root Induction in Arabidopsis
   1. Lateral Root Inhibition (NPA Treatment)
      1. Prepare growth medium for in vitro growth. The medium contains half strength Murashige and Skoog salt mixture¹⁵, 0.1 g/L myo-inositol, 10 g/L sucrose, and is buffered with 0.5 g/L 2-(N-morpholino)ethanesulfonic acid (MES).
      2. Adjust the pH to 5.7 with 1 M KOH. Add 8 g/L of plant tissue agar and autoclave the medium for 20 min at 121 °C.
      3. Prepare a 25 mM stock solution of NPA in dimethylsulfoxide (DMSO) (CAUTION: Use gloves). In a laminar air flow cabinet, cool the autoclaved medium down to approximately 65 °C (which is the temperature at which the bottle can be held by hand), and add 25 mM NPA stock solution to obtain a final concentration of 10 µM NPA.
      4. Homogenize upon addition by gently shaking the bottle for 10 sec. Pour 50 ml of medium per square petri dish (12 cm x 12 cm).
         Note: In case of large scale experiment, apply a nylon mesh (20 µm) to ensure easy transfer. Prepare a mesh (9 cm x 9 cm) for autoclaving (Figure 1A). When autoclaved, apply the mesh to the growth medium using sterile tweezers (Figure 1B). Gently push the mesh to the growth medium using a sterilized drigalsky to make sure it is in good contact with the growth medium (Figure 1B) (CAUTION: Work in sterile conditions). 
      5. Sow the seeds on the NPA-plates.
         Note: In case a large scale experiment is planned, sow 2 dense and well-aligned rows of 50 seeds to facilitate the sampling (see 1.2.2.3).
         1. In case of liquid sterilization, sow the seeds on NPA-plates using a 200 µl pipet. Cut off 3 - 4 mm of the end of the pipetting tips in sterile conditions (or before sterilization of the tips) in order to be able to pipet the seeds. Pipet up and down a couple of times to re-suspend the seeds, then pipet approximately 150 µl sterile water with 10 - 20 seeds and wait till the seeds sediment at the bottom of the tip. (CAUTION: Work in sterile conditions)
            Note: When touching the agar or the mesh gently with the tip, a drop of water with a seed will be released from it (Figure 1C).
         2. In case of gas sterilization, sow the seeds using autoclaved toothpicks.Pinch the toothpick into the agar before taking the seeds to ensure a sticky surface. Pick up the seeds one by one using the toothpick and distribute the seeds on the plates (Figure 1D). Alternatively, add sterile water to the seeds, and sow as described in 1.2.1.5.1 (CAUTION: Work in sterile conditions).
      6. Seal the plates with breathable adhesive tape and store them plates at 4 °C in the dark for at least 2 days and maximum one week. This is needed for stratification and ensures synchronized and more efficient germination.
         Note: Alternatively, seeds can be stratified before sowing, immersed in distilled water in micro-centrifuge tubes, which requires less refrigerator space. In this case, store the tubes for at least one week at 4 °C, and move the plates to the growth cabinet immediately after sowing (see 1.2.1.7).
         DAY 3
      7. Move the plates to the growth cabinet (continuous light (110 µE m^-2 s^-1), 21 °C) for 5 days (2 days of germination and 3 days of growth) in a nearly vertical position (80 to 90 °) to ensure root growth on, and not in the medium.
         DAY 8 
   2. Lateral Root Induction (NAA Treatment)
      1. Prepare the same growth medium as described in 1.2.1.1 and 1.2.1.2. Prepare a 50 mM stock solution of NAA in DMSO (CAUTION: Use gloves). Cool the autoclaved medium down to 65 °C and add NAA solution to the medium to obtain a final concentration of 10 µM NAA (CAUTION: Work in sterile conditions).
         1. Homogenize upon addition by gently shaking the bottle for 10 sec. Pour 50 ml of medium per square petri dish (12 cm x 12 cm).
      2. Transfer the seedlings from the plates containing growth medium supplemented with 10 µM NPA to those supplemented with 10 µM NAA.
         1. Hook the arms of curved tweezers under the cotyledons of the seedling and gently remove it from the plate. Only transfer seedlings of which the roots have grown entirely in contact with the NPA-growth medium and preferably downwards.
         2. Skim the root over the NAA-containing growth medium surface over a small distance. Make sure that the plant roots are in good contact with the growth medium. If needed, press the root gently to the growth medium surface with the tweezers.
            Note: In case of a large-scale experiment, remove all seedlings that are not in contact with the mesh and transfer by lifting the mesh at the two upper corners with tweezers. Make sure the mesh is in good contact with the NAA-containing medium by skimming the mesh over the agar surface over a small distance (Figure 1E). 
      3. Seal the new plates with breathable tape and place them in the growth cabinet (continuous light (110 µE m^-2 s^-1), 21 °C) in a vertical position for the desired time after induction until sampling.
         Note: In case of a large-scale experiment, use a scalpel to cut the root segments all at once directly on the mesh and sample them by gently scraping the surface of the mesh.  

2. LRIS Maize Protocol

1. Stratification of Maize Kernels
   1. Incubate the maize kernels at 4 °C in the dark for at least 1 week.
      Note: This protocol has been developed using the B73 maize inbred line.
2. Sterilization of Maize Kernels
   DAY 1
   Note: Although maize seedlings do not have to be grown in sterile conditions, it is recommended to sterilize the maize kernels and work with sterile material to prevent fungal growth in the paper roll system.
   1. Put the necessary number of maize kernels in a sterilized glass beaker.
   2. Add 100 ml of sterilization solution (6% NaOCl in water) (CAUTION: Use fume hood).
   3. Add a magnetic stir bar and place the beaker on a magnetic stirrer at low stirring speed (approximately 250 rpm) at RT for 5 min.
   4. Rinse five times for 5 min by replacing the solution with 100 ml of fresh sterile water.
3.  Sowing Maize Kernels
   1. Put on gloves to reduce the risk of contamination, and take a roll of paper towels.
   2. Tear off two stretches of hand towel paper with a total length of two sheets (approximately 92 cm x 24 cm) and place them on top of each other on a clean surface (Figure 2A).
   3. Fold the sheets double over the length (approximately 92 cm x 12 cm) (Figure 2A).
   4. Use tweezers to distribute 10 kernels at approximately 2 cm from the top over the entire length of the paper, while keeping an interspace of 8 cm between each kernel and 8 cm free at both ends (Figure 2B). Make sure the radicle of the kernel is facing down and toward the paper (Figure 2B).
   5. Gently roll the paper over the length, while keeping the seeds in place (Figure 2B). To facilitate the rolling, it is optional to first spray the paper with sterile water.
   6. Put the paper rolls in sterilized glass tubes of approximately 6 cm diameter and 14 cm height (e.g., 250 ml centrifuge tubes) and place them in a rack (Figure 2C).
4. Lateral Root Induction in Maize
   1. Prepare a 50 mM NPA stock solution (dissolved in DMSO) (CAUTION: Use gloves).
   2. For each tube, make a 50 µM NPA solution by adding 125 µl from the 50 mM NPA stock solution to 125 ml sterile water in a sterilized glass beaker.
   3. Mix well and pour 125 ml of the 50 µM NPA solution over the paper roll in the tube. The paper roll will absorb the solution and become completely soaked.
      Note: When using paper rolls and tubes with other dimensions, adjust the volume accordingly. The liquid should reach halfway the tube to ensure sufficient uptake.
   4. Place the paper roll system in a growth cabinet for three days (27 °C, continuous light, 70% relative humidity). Make sure that the paper rolls remain soaked by adding extra 50 µM NPA solution, since the solution evaporates over time (Figure 2D).
      DAY 4
   5. Prepare a 50 mM NAA stock solution (dissolved in DMSO) (CAUTION: Use gloves).
   6. For each tube, prepare a 50 µM NAA solution by adding 125 µl from the 50 mM NAA stock to 125 ml sterile water in a sterilized glass beaker.
   7. Gently squeeze out most of the remaining NPA solution from the paper rolls and place them in sterile water for 5 min to wash it out (CAUTION: Use gloves). Gently squeeze out most of the water from the paper rolls. Repeat this washing step three times.
   8. Mix well and pour the 50 µM NAA solution over the paper rolls in the tubes. Make sure they are completely soaked.
   9. Place the paper roll system in the growth cabinet (27 °C, continuous light, 70% relative humidity) for the desired duration after induction.
5. Lateral Root Induction in Adventitious Crown Roots of Maize
   Note: The LRIS as described above induces lateral roots in the primary root. However, in maize and other monocotyledons, the primary root and the embryonic seminal roots, together with their lateral roots, are mainly important during seedling development. At a later stage in plant growth, the post-embryonic adventitious roots arise on the stem and also develop lateral roots to form a new root system¹⁶. The LRIS can also be used to induce lateral roots on these post-embryonic adventitious crown roots by following some minor adjustments to the LRIS on the embryonic primary root.
   1. To make the paper rolls, create a sandwich of papers with respectively two layers of hand towel paper on the outer side, and two layers of germination paper within. Place the sterilized kernels (see steps 2.1 to 2.2) in between the two sheets of germination paper. Germination paper, compared to hand towel paper, will be less prone to be penetrated by root hairs and lateral roots, which will make the opening of the paper rolls easier later on. On the other hand, the two external layers of hand towel paper will facilitate absorption of the liquid.
   2. Insert the rolls in 700 ml glass tubes and pour sterile water without NPA over them. Make sure they are completely soaked.
   3. Germinate the sterilized kernels in the growth cabinet (see step 2.4.9).
   4. Grow the seedlings till the adventitious crown roots start to emerge (6 days for B73). Make sure the paper rolls are kept wet at all times by adding sterile water when necessary.
   5. Transfer to a 25 µM NPA solution for 4 days (see steps 2.4.1 to 2.4.4), followed by a similar induction of lateral root initiation by replacing the NPA solution with a 50 µM NAA solution after a washing step (see step 2.4.5 to 2.4.9).

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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 ...

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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 ...

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Materials

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