An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

Summary

The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

Abstract

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.

Introduction

The plant stoma is an essential organ for gas exchange for photosynthesis and transpiration, and stomatal movement is accomplished by significant changes in guard cells through ion-driven uptake and release of water. Under a microscope, we can observe a spaced distribution pattern of stomata on the surfaces of leaves and stems. This spaced distribution of stomata is considered to help stomatal movement, which is regulated by ion and water exchange between guard cells and neighboring epidermal cells^1,2. Experimental induction systems for clustered stomata are useful for investigating the importance of the spaced distribution of stomata.

It has been reported that spatial clustering of stomata can be induced by genetic modification of key genes for guard cell differentiation^3,4 or treatment with a chemical compound⁵. We also reported that immersion treatment with a medium solution supplemented with sugars including sucrose, glucose, and fructose caused stomatal clustering in cotyledons of Arabidopsis thaliana seedlings⁶. Reduced callose in new cell walls separating meristemoids and epidermal cells was observed in the sucrose-treated cotyledon epidermis, suggesting that sucrose solution immersion treatment negatively affects the cell wall, which prevents the leakage and ectopic action of key gene products for guard cell differentiation (e.g. transcription factors) towards adjacent epidermal cells⁶. A similar mechanism was suggested from studies on gsl8/chor mutants^7,8. Our experimental system for reproducible induction of clustered stomata using sucrose-containing medium solution is quite easy and cheap. It can also be used to investigate intracellular structures such as organelles and the cytoskeleton in the clustered guard cells when applied to transgenic lines expressing fluorescent markers that label intracellular structures^9,10.

Protocol

1. Preparation of 3% Sucrose-containing 1/2 Murashige-Skoog Medium Solution

1. Add 1.1 g of Murashige-Skoog medium salts and 15 g of sucrose to a beaker.
2. Add 490 mL of distilled water and mix well using a stir bar.
3. Adjust the pH to 5.8 using KOH.
4. Dilute to 500 mL with distilled water and transfer the solution into a medium bottle.
5. Sterilize the solution by autoclaving (121 °C, 20 min). If not used immediately, this solution may be kept at 4 °C after sterilization.

2. Induction of Clustered Stomata by Sucrose-Containing Medium Solution Immersion Treatment

1. Sterilize the seeds.
   1. Prepare the sterilization solution by adding 500 µL of 5% active chlorine NaClO solution and 1 µL of 10% Triton X-100 to 500 µL sterile water.
   2. Place ca. 50 transgenic A. thaliana seeds carrying a fluorescent marker such as CT-GFP¹¹ or GFP-TUB6¹² into a 1.5-mL tube.
   3. Add 1 mL of 70% ethanol solution and mix well by inverting five times. Leave for 1 min.
   4. The seeds will sink to the bottom of the tube. On a clean bench, gently remove the 70% ethanol using a micropipette, and add 1 mL of sterilization solution. Mix well by inverting five times and leave for 5 min.
   5. Wash the seeds. Still working under aseptic conditions on a clean bench, gently remove the solution using a micropipette, and add 1 mL of sterile water. Repeat this step five times.
2. Add 1.5 mL of sterilized 3% sucrose-containing 1/2 Murashige-Skoog medium solution to each well of a 24-well plate on a clean bench.
3. Add two sterilized seeds into each well. Tape the lid onto the 24-well plate using two layers of parafilm.
4. Transfer the 24-well plate to a growth chamber set at 23.5 °C with a 12-h/12-h light-dark cycle using 100 µmol m⁻² s⁻¹ white light and incubate for 14 days.

3. Microscopic Observation of Clustered Stomata

1. Place 30 µL of 3% sucrose-containing 1/2 Murashige-Skoog medium solution from a well of the 24-well plate onto the center of a glass slide (size: 76 × 26 mm, thickness: 1.0-1.2 mm).
2. Remove a cotyledon from a 14-day-old seedling using dissecting scissors. Float the cotyledon with the observation side facing up on the solution drop.
3. Prepare the cotyledon specimen according to our previous method¹³. Essentially, place 30 µL of the solution on the center of a cover glass (size: 18 × 18 mm, thickness: 0.12-0.17 mm). Turn the cover glass upside down and place it on the cotyledon gently. Wipe off excess buffer using a lint-free tissue.
4. Set a specimen on the stage of a confocal laser microscope and select clustered guard cells for observation using bright field illumination.
5. Acquire confocal images of fluorescently labelled intracellular structures according to the microscope manufacturer's instructions.

Results

Here, the protocol for a simple method of inducing stomatal clustering with sucrose-containing medium solution in A. thaliana seedlings has been presented. The clustered guard cells grown in sucrose-containing medium solution (Figure 1B) have larger chloroplasts than guard cells grown in sucrose-free control conditions (Figure 1A). The enlargement of chloroplasts was confirmed with CT-GF...

Discussion

We have presented protocols for induction of clustered stomata in A. thaliana seedlings by immersion treatment with a sucrose-containing medium solution. As shown here, this method is very simple and requires no specialized skill but can efficiently induce clustered stomata. More than 45% of guard cells are clustered with 3% sucrose-containing medium solution (mean values of more than 20 independent observations)⁶. Moreover, this experimental system can be directly applied to transgenic o...

Materials

Name	Company	Catalog Number	Comments
24-well plate	Sumitomo Bakelite	MS-0824R
488 nm laser	Furukawa Denko	HPU-50101-PFS2
488 nm laser	Olympus	Sapphire488-20/O
510 nm long-pass filter	Olympus	BA510IF
524 - 546 nm band-pass filter	Semrock	FF01-535/22-25
530 nm short-pass filter	Olympus	BA530RIF
561 nm laser	CVI Melles Griot	85-YCA-025-040
604 - 644 nm band-pass filter	Semrock	FF01-624/40-25
Confocal laser scanning head	Yokogawa	CSU10
Confocal laser scanning head	Olympus	FV300
Cooled CCD camera	Photometrics	CoolSNAP HQ2
Image acquisition software	Molecular Devices	MetaMorph version 7.8.2.0
Image acquisition software	Olympus	FLUOVIEW v5.0
Immersion oil	Olympus	Immersion Oil Type-F	ne = 1.518 (23 degrees)
Inverted microscope	Olympus	IX-70
Inverted microscope	Olympus	IX-71
Murashige and Skoog Plant Salt Mixture	FUJIFILM Wako Pure Chemical Corporation	392-00591	Murashige T and Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15(3), 473-497.
Objective lens	Olympus	UPlanApo 100x / 1.35 NA Oil Iris 1.35	NA = 1.35
Objective lens	Olympus	UPlanAPO 40x / 0.85 NA	NA = 0.85
Sucrose	FUJIFILM Wako Pure Chemical Corporation	196-00015