12.2 Protonema development

Yuji Hiwatashi and Yoshikatsu Sato

Introduction

This protocol will be useful to analyze protonema development such as growth, cell division, the number of chloroplast in P. patens.

Marerials: White light grown protonemata in glass-bottom dish

(1) Pre-culture

Overlay BCDATG medium on a 35 mm glass-bottom dish (IWAKI 3910-039: a 27 mm diameter opening in the center of a dish,

Inoculate ~7-day-old protonema tissue on the center region of BCDATG medium and seal the glass-bottom dish with surgical tape.

Incubate the dish under white light (60 µmolm-2sec-1) at 25˚C for 4~7 days.　

(2) Observation

(2-1) Analysis of cell length and width

We measure length and width of a subapical cell or a second subapical cell.

Capture an image of a subapical cell or a second subapical cell under a microscope.

Measure length and width of these cells using ImageJ.

(2-2) Analysis of growth velocity and cell division by time-lapse observation

Time-lapse image acquiring enables us to analyze the growth velocity and process of cell division as well as cell length and width.

1. Keep room temperature at 25˚C.

2. Place the dish on the stage of an inverted microscope.

3. Seek a protonemal apical cell just before cell division and focus on its nucleus. The apical cell before cell division is much longer than its neighboring subapical cell and its cytoplasm is localized at the apical end. One of the signs of mitosis is a transition of nuclear shape. Just before entering prophase, a nucleus becomes spherical rather than oval.

4. Carry out time-lapse observation. M-phase ends within ~40 min in this condition, so take an image at every 60 sec. Excitation light intensity should be weak as far as possible especially when GFP signals are observed. Strong light inhibits cell division and fades GFP fluorescence.

(2-3) The numbers of chloroplasts

The procedure for counting the numbers of chloroplasts was described in the previous paper (Hayashida et al. 2005). The numbers of chloroplasts in subapical cells of chloronemata can be counted under a light microscope. Average numbers in a chloronema cell of wild type are about 50.

Marerials: Protonemata grown under unilateral red light

Protonemata grown under unilateral red light were used for analyses of chloroplast movement (Kadota et al. 2000). The resultant filaments are also tractable system for analyses of protonema development for the following reason. Firstly, many protonemata are generated and phototoropic growth towards red light aligned resultant filaments parallel each other, facilitating us to search target cells. Secondly, protonemata grow uniformly without branches, enabling us to collect populations easily. Thirdly, side branch initiation is induced by the subsequent white light irradiation, allowing us to analyze the stem cell formation such as study of the cell cycle re-entry.

(1) Pre-culture

1. Protonemal cells are sub-cultureed on cellophane-overlaid BCDAT plates under continuous white light (30-60 µmol m-2 sec-1) at 25˚C every 5~7 days.

2. Small amount of 5~7-day-old protonema cells are inoculated on BCDAT plates (35 mm-diameter dish) and covered with a sterile cover slip (18 x18 mm).

3. The protonema tissues are cultured under unilateral red light (3~5 µmol m-2 sec-1) at 25˚C for 7 days.　 Red light can be obtained from fluorescent tube filtered through a red plastic sheet (We use shikolite 102; Mitsubishi Rayon, Japan, http://www.mrc.co.jp/shinkolite/index.html)

(2) Observation

(2-1) Protonema growth

1. Capture images of overall protonemal filaments using a stereomicroscope.

2. Compare the length from inoculated cells to apical cells of protonemal filaments.

Apical protonema growth shows a typical tip growth, and a daughter cell divided from the apical cell does not growth. Therefore, the difference of the protonema filament length only depends on the rate of tip growth. We can detect easily the growth defect.

(2-2) Cell length and width

We measure length and width of a subapical cell or a second subapical cell.

Capture an image of a subapical cell or a second subapical cell under a microscope.

Measure length and width of these cells using ImageJ.

(2-3) Analysis of cell division by time-lapse observation

For this purpose, time-lapse observation is performed.

1. Remove a cover slip from the plate and cut an agar block containing protonema cell from the solid medium with a scalpel. Turn the block up-side down and place it into 35 mm glass-bottom dish (IWAKI 3910-039: a 27 mm diameter opening in the center of a dish, http://www.atgc.co.jp/div/rika/hbine/index_e.html) as protonema cell are touched on the bottom of the dish.　 Seal the dish with parafilm.

2. Place the dish on a stage of an inverted microscope. While you observe, room temperature should be set at 25˚C.

3. Seek protonemal apical cell just before cell division and focus a nucleus. An apical cell before division is highly elongated and its cytoplasm is localized to apical side of a cell. One of the signs of mitosis is transition of a nuclear shape. Just before entering prophase, a nucleus will be a spherical shape rather than an oval shape.

3. Start the time-lapse image acquiring. M-phase will finish ~40 min in this condition. We usually acquire an image every 60 sec. If you examine the dynamics of GFP-fusion protein, you should not irradiate strong excitation light to the cell for preventing from not only fluorescence fading but also damaging cellular function.

(2-4) The numbers of chloroplasts

When you count the numbers of chloroplasts in a white light grown cell under a light microscope, crossover of chloroplasts often makes you difficult to numerate them accurately. One solution is counting them from the LSM Z-sectioning images. However, another soft solution is counting them in a red light grown cell. Cell length of red light grown protonema (about 120 µm) is longer than that of white light grown one (about 80 µm) and the size of chloroplasts in red light grown cell is smaller than that in white light grown one, posses, facilitating us to count the numbers of chloroplasts.

(2-5) Quantitative analysis from time-lapse data

Time-lapse data tells us dynamic properties of cells. Here, we will introduce a representative method for quantitative analysis from time-lapse data. The method is ‘kymograph’. Kymograph shows a time space plot

The left image is the original kymograph data of protonemal growth. We can get the following properties of the protonema cell from one time-lapse experiment.

1) Changes of growth velocity

2) Nuclear behaviors

3) Chloroplast behaviors

4) Cell cycle period

5) Cell length

We usually use Metamorph software at http://www.moleculardevices.com for kymograph analysis. You can do kymograph analysis using freeware, Kymograph Plugin for ImageJ at http://www.embl.de/eamnet/html/body_kymograph.html.

Reference

1) Kadota A, Sato Y, Wada, M. (2000) Planta 210, 932-937

2) Hayashida et al. (2005) Plant Biology 7, 300-306