Quantification of metabolic activity of cultured plant cells by vital staining with fluorescein diacetate

Accepted Manuscript Quantification of metabolic activity of cultured plant cells by vital staining with fluorescein diacetate Naoko Saruyama, Yurina Sakakura, Tomoya Asano, Takumi Nishiuchi, Hamako Sasamoto, Hiroaki Kodama PII: DOI: Reference:

S0003-2697(13)00281-9 http://dx.doi.org/10.1016/j.ab.2013.06.005 YABIO 11384

To appear in:

Analytical Biochemistry

Received Date: Revised Date: Accepted Date:

30 March 2013 1 June 2013 3 June 2013

Please cite this article as: N. Saruyama, Y. Sakakura, T. Asano, T. Nishiuchi, H. Sasamoto, H. Kodama, Quantification of metabolic activity of cultured plant cells by vital staining with fluorescein diacetate, Analytical Biochemistry (2013), doi: http://dx.doi.org/10.1016/j.ab.2013.06.005

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Quantification of metabolic activity of cultured plant cells by vital staining with fluorescein

2

diacetate

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Naoko Saruyama1, Yurina Sakakura2, Tomoya Asano3, Takumi Nishiuchi3, Hamako Sasamoto4,

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Hiroaki Kodama2

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1

Graduate School of Horticulture, Chiba University, 648 Matsudo, Chiba 271-8510, Japan

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2

Graduate School of Advanced Integration Science, Chiba University, 1-33 Yayoi-cho, Chiba 263-

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8522, Japan 3

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Division of Functional Genomics, Advanced Science Research Center, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan

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Faculty of Environment and Information Sciences, Yokohama National University, Yokohama 240-8501, Japan

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Correspondence: Dr. Hiroaki Kodama

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e-mail: [email protected]

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Tel/Fax: +81-43-290-3942

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Short title: Quantification of plant cellular activity

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Subject category: Cell Biology

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1

Abstract

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The metabolic activity of suspension cultures of Sonneratia alba cells was quantified by

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measurement of the hydrolysis of fluorescein diacetate (FDA). FDA is incorporated into live cells

4

and is converted into fluorescein by cellular hydrolysis. Aliquots (0.1–0.75 g) of S. alba cells were

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incubated with FDA at a final concentration of 222 µg/mL suspension for 60 min. Hydrolysis was

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stopped, and fluorescein was extracted by the addition of acetone and quantified by measurement of

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absorbance at 490 nm. Fluorescein was produced linearly with time and cell weight. Cells of S. alba

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are halophilic and proliferated well in medium containing 50 and 100 mM NaCl. Cells grown in

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medium containing 100 mM NaCl showed 2- to 3-fold higher FDA hydrolysis activity than those

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grown in NaCl-free medium. When S. alba cells grown in medium supplemented with 50 mM NaCl

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were transferred to fresh medium containing 100 mM mannitol, cellular FDA hydrolysis activity

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was downregulated after 4 d of culture, indicating that the moderately halophilic S. alba cells were

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sensitive to osmotic stress. Quantification of cellular metabolic activity via the in vivo FDA

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hydrolysis assay provides a simple and rapid method for the determination of cellular activity under

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differing culture conditions.

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Keywords

Cell viability・esterase・fluorescein diacetate・osmotic stress・salt tolerance

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1

Introduction

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The plantation and preservation of forests has enormous environmental value, but the slow-growing

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nature of trees is one barrier to meeting current demands [1]. The application of plant biotechnology

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for tree improvement has long been desired to sustainably fulfill the demand for wood. The in vitro

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culture of woody plants is time-consuming and laborious because of their slow growth. The

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optimization of culture conditions is achieved by the measurement of cell growth parameters, such

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as fresh weight (FW), dry weight (DW), settled cell volume (SCV) and/or packed cell volume

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(PCV) after prolonged culture periods. Cultured woody plant cells are often recalcitrant to

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enzymatic cell wall hydrolysis, and direct determination of cell number is generally difficult.

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Because these physiological traits have often been observed for woody plant cells, the rapid and

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simple evaluation of cell viability would be a valuable basic technique for the development of tree

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biotechnology.

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Vital staining with fluorescein diacetate (FDA) has long been used for the detection of viable

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plant cells [2]. A nonpolar substrate, FDA, is incorporated into plant cells, where it is hydrolyzed by

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esterases to produce a polar product, fluorescein. Fluorescein is retained intracellularly because it is

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weakly transported through the plasma membrane [3]. After staining with FDA, fluorescein-

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positive viable cells can be visualized by fluorescence microscopy [4,5]. FDA has also been used

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for the determination of microbial activity in soil and litter because microorganisms also hydrolyze

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FDA, and microbial activity is evaluated by measuring the absorbance of fluorescein [6].

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Kawana and Sasamoto [7] established a suspension culture of a mangrove plant, Sonneratia alba.

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The suspension-cultured S. alba cells were halophilic, and 25 to 100 mM NaCl stimulated growth in

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comparison to the NaCl-free culture. In addition, protoplasts prepared from cotyledons of S. alba

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also showed a halophilic response to NaCl, KCl and MgCl2 in a concentration range of 10 mM to

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50 mM, suggesting that S. alba has an intrinsic halophilic nature [8]. These halophilic properties of

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S. alba cells were evaluated by measuring cell growth after prolonged culture. An understanding of

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the halophilic phenotype of S. alba might be valuable for the development of salt-tolerant plants. 3

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Here, we report a rapid and simple method for determining the cell viability and/or metabolic

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activity of suspension cultures of S. alba cells by vital staining with FDA. We focused on the

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effects of stress factors on the growth of S. alba cells. The FDA hydrolysis assay was investigated

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as a potential measure of cell viability and/or cellular metabolic activity, and we asked whether

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quantified values of FDA hydrolysis activity correlated with cell growth in the S. alba cell culture.

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The halophilic properties and cellular responses to osmotic stress were compared with the FDA

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hydrolysis activity. The enhanced growth in the presence of NaCl was associated with a high

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cellular FDA hydrolysis activity. The inhibitory effects of osmotic stress on cell growth were in

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agreement with the decrease in FDA hydrolysis activity. These results indicate that quantification of

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FDA hydrolysis activity is useful for the rapid assessment of cellular responses to medium

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components and stress factors.

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Materials and Methods

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Plant material

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Suspension cultures of S. alba were induced from cotyledons as previously described [9]. S. alba

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cells were maintained at 30°C in Murashige-Skoog (MS) medium [10] supplemented with 0.1 µM

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2,4-dichlorophenoxyacetic acid, and cultures were renewed every three weeks by transferring 3 mL

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of cells into 15 mL of fresh medium. S. alba cells were maintained on a rotary shaker (NR-20,

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TAITEC, Japan) at 100 rpm in the dark. Aliquots of cells were separately maintained in MS

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medium containing 0 mM, 50 mM or 100 mM NaCl for more than one year. Growth was

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determined in four independent flasks. The suspension of each flask was filtered through Miracloth

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for the determination of FW. For the determination of SCV, the suspension of S. alba cells was

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transferred to centrifuge tubes and left for 60 min before the sedimented cell volume was

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determined.

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Exposure to osmotic stress 4

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S. alba cells that were maintained in MS medium containing 50 mM NaCl or in NaCl-free MS

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medium were transferred to fresh medium supplemented with 100 mM mannitol. During mannitol

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treatment, cells were cultivated in NaCl-free conditions for 4 d and then subjected to assays for in

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vivo FDA hydrolysis and esterase activities. The effect of mannitol on cell growth was determined

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by measurement of SCV after a prolonged culture period.

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Colorimetric quantification of in vivo FDA hydrolysis activity

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FDA hydrolysis was determined according to a modified protocol for the determination of total

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microbial activity [6]. After optimizing the assay conditions, the assay was performed as follows:

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FDA was dissolved in acetone at a concentration of 2 mg/mL. Cells were collected on Miracloth by

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vacuum filtration, and 0.5 g cells were resuspended in 4 mL of fresh MS medium. The resuspended

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cultures were incubated at 30°C on a rotary shaker at 100 rpm for 60 min and the assay was

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initiated by the addition of 0.5 mL FDA solution. Hydrolysis of FDA was stopped, and cells were

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lysed by the addition of 2 mL acetone. Cell debris was removed by centrifugation at 10,000 g for 5

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min, and the fluorescein concentration in the supernatant was measured by absorbance at 490 nm.

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Esterase activity in the protein extract

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In addition to FDA, 1-naphthylacetate (1-NA) and 2-naphthylacetate (2-NA) were used as broad

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spectrum substrates for esterases [11]. Cells were collected on Miracloth by vacuum filtration and

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immediately frozen at -80°C. Approximately 0.1 g of frozen cells was homogenized with 1.5 mL of

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0.1 M Tris-HCl, pH 8.0, using a mortar and pestle. Cell debris was eliminated from the homogenate

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by centrifugation at 10,000 g for 30 min, and the supernatant was centrifuged again at 10,000 g for

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30 min. The supernatant was subsequently subjected to the esterase activity assay. The esterase

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activity using 1- and 2-NA as substrates was determined according to the procedure of Balen et al.

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[12]. The reaction solution consisted of 0.35 mL of 50 mM Tris-HCl, pH 7.5, and 15 µL of 0.1 M 1-

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NA or 2-NA dissolved in absolute methanol. The reaction was started by the addition of 0.1 mL of 5

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crude extract. When FDA was used as the substrate, the reaction solution contained 0.4 mL Tris-

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HCl, pH 7.5, and 50 µL of crude extract, and the reaction was started by the addition of 25 µL of 2

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mg/mL FDA dissolved in acetone. The esterase activity was determined spectrophotometrically by

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measuring the increase in absorbance at 322 nm (for 1-naphthol), 313 nm (for 2-naphthol) and 490

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nm (for fluorescein).

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Results

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Quantification of cellular metabolic activity by FDA hydrolysis

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The S. alba suspension cultures were maintained in MS medium with 0 mM, 50 mM or 100 mM

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NaCl for more than one year. The growth cycle of S. alba cells was determined by the measurement

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of SCV and consisted of a short lag phase, a logarithmic phase of 12 d and a stationary phase that

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was reached after approximately 15 d of subculture (Fig. 1A). Addition of NaCl to the medium

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stimulated cell growth as previously reported [7], and the FW of cells at the stationary phase (after

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21 d of subculture) was higher in the cultures containing 50 mM or 100 mM NaCl (Fig. 1B). We

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asked whether suspension cultures grown in the presence of 50 or 100 mM NaCl consisted of

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metabolically active cells in comparison with those grown in NaCl-free medium. Fluorescence

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microscopy showed that 16-d-old S. alba cells were well stained with FDA, irrespective of the NaCl

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concentration of the medium (Fig. 1C). In fact, no distinguishable difference in the fluorescence

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patterns at 10 min and 30 min after staining was observed between cells cultured in NaCl-free

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medium or cells grown with 100 mM NaCl (Fig. S1).

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We then assessed cellular viability by quantifying the products of FDA hydrolysis. The FDA

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hydrolysis reaction was used as a possible measure of cell viability and/or cellular metabolic

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activity. The basic procedure for quantification of cell viability was developed in a previous study

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[6] to determine soil bacterial activity. Although fluorescein is a fluorescent substance, this pigment

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absorbs at 490 nm. FDA does not show any absorbance at 490 nm (Fig. S2). We therefore

6

1

determined the amount of fluorescein produced through the colorimetric measurement of

2

absorbance at 490 nm.

3

First FDA was directly added to the 21-d-old suspension culture. Unexpectedly, fluorescein was

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detected in the cell-free supernatant of the culture medium (Fig. 2A). Thus, S. alba cells may

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secrete esterase-like enzymes, or the esterase-like activity in the medium could come from lysed

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cells. At present, we have not identified the responsible proteins. To exclude FDA hydrolysis

7

activity in the medium, culture medium was replaced with fresh medium, and cellular viability was

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assessed (see Materials and Methods).

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We determined the optimum FDA concentration and incubation time as well as the relationship

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between the amount of cells and FDA hydrolysis. FDA is dissolved in acetone. Because a high

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concentration of acetone would affect cellular activity, the final concentration of acetone in the

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assay solution should be less than 15% (v/v) (Fig. S3). The 21-d-old S. alba cells grown in MS

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medium containing 50 mM NaCl were collected by filtration, and aliquots of cells were

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resuspended in fresh culture medium containing 50 mM NaCl. FDA was added at the

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concentrations indicated in Figure 2B, and cells were incubated for 60 min. The hydrolysis reaction

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was terminated, and cells were lysed by the addition of excess acetone. The amount of fluorescein

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increased approximately linearly with the increase in FDA until a final concentration of 111 µg/mL

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suspension culture. The absorbance at 490 nm with the given amount of cellular material was

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negligible, and the assay without FDA showed a low background absorbance (Fig. 2B). We added

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FDA at a final concentration of 222 µg/mL in subsequent experiments. Next, we determined the

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time course of fluorescein production. The amount of fluorescein increased linearly with time for 60

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min (Fig. 2C). Finally, we determined the effect of the amount of cells on the FDA hydrolysis

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reaction; fluorescein was produced linearly when 0.10 to 0.75 g FW of cells was incubated with 222

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µg/mL FDA for 60 min (Fig. 2D). Due to the enhanced growth in the presence of NaCl,

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approximately a 3-fold higher FDA hydrolysis activity was observed in cells grown with 100 mM

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NaCl than in cells cultured in NaCl-free medium. In subsequent experiments, 0.5 g of cells was

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used for the in vivo FDA hydrolysis assay.

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Osmotic response of S. alba cells

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Exposure to high salt concentration imposes osmotic stress as well as ionic stress. We therefore

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addressed whether S. alba cells grown in the presence of 50 mM NaCl had an increased tolerance

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against osmotic stress. The 21-d-old cells grown in MS medium containing 50 mM NaCl were

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transferred to fresh MS medium containing 100 mM mannitol instead of NaCl. In parallel, the 21-d-

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old cells grown in NaCl-free MS medium were transferred to fresh MS medium with 100 mM

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mannitol, and FW and in vivo FDA hydrolysis activity were determined for both cultures 4 d after

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transfer (Fig. 3A). The growth of cells was nearly identical between the cultures tested. However,

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FDA hydrolysis activity of cells treated with mannitol decreased by approximately 60%. An

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equivalent decrease in FDA hydrolysis activity by mannitol treatment was observed in the cells that

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were cultured in the NaCl-free and 50 mM NaCl-containing MS medium. The inhibitory effect of

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mannitol on cell growth manifested 11 d after transfer. As expected from the result of the in vivo

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FDA hydrolysis experiments, the SCV of the 11-d-old cells grown with mannitol decreased by

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approximately 70% in comparison with that of cells grown with 50 mM NaCl (Fig. 3B). This result

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indicated that S. alba cells grown with 50 mM NaCl had a low level of tolerance against 100 mM

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mannitol.

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The amount of fluorescein produced depends upon the intracellular FDA level and also upon the

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level of esterase activity. We then determined the enzymatic activity of esterases in crude protein

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extracts by using FDA and two other broad spectrum substrates, 1-NA and 2-NA [11,12]. Total

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protein was extracted from the 4-d-old cells grown with 100 mM mannitol or 50 mM NaCl. The

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enzymatic hydrolysis activity in the protein extracts decreased by 18% (1-NA), 25% (2-NA) and

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72% (FDA) in the cultures with mannitol (Fig. 3C). This result indicated that the in vivo FDA

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hydrolysis activity correlated with the in vitro enzymatic activity of FDA hydrolysis. Because cell 8

1

growth in the presence of mannitol decreased by approximately 70% (Fig. 3B), cellular growth

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activity can be estimated from the in vivo FDA hydrolysis assay.

3 4

Discussion

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Here, we report the development of an assay for the rapid quantification of cellular metabolic

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activity and/or cell viability via vital staining with FDA. Based on experiments to determine the

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substrate FDA levels, cell volume and time course, an optimized FDA hydrolysis assay is described

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(see Materials and Methods). The scale of the assay can be reduced if the amount of cultured cells is

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limited; we often determined FDA hydrolysis using 100 mg FW of cells. Cells of S. alba are

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moderately halophilic because cultures supplemented with 50 mM and 100 mM NaCl produced

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more cell mass than NaCl-free cultures (Fig. 1B) [7]. Accordingly, FDA hydrolysis activity was

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approximately 2- to 3-fold higher in cells grown with 100 mM NaCl than in cells in NaCl-free

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conditions (Figs. 2C and 2D). It was difficult to evaluate differences in cellular metabolic activity

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microscopically because cells from cultures with and without NaCl showed similar fluorescence

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after FDA staining (Fig. 1C, Fig. S1). Therefore, differences in fluorescein production among S.

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alba cell cultures are most likely due to differences in cellular esterase activities and not to

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differences in the proportion of viable cells in the total cell population.

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S. alba cells are moderately tolerant to sodium ion stress but are sensitive to osmotic stress. After

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prolonged culture with mannitol, decreased cell growth was observed (Fig. 3B). In contrast, the

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reduction in the FDA-hydrolyzing activity was observed within a few days after transfer to

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mannitol-containing medium. These results indicated that mannitol treatment suppressed cellular

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metabolic activity within a few days, and the FDA hydrolysis activity was concomitantly decreased.

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When 1-NA and 2-NA were used as substrates, the esterase activities in the mannitol-treated

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cells were reduced to a lesser degree than the in vitro FDA hydrolysis activity. There are two

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possible explanations. One is that FDA-hydrolyzing enzymes are different from the esterases that

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can catalyze the hydrolysis of 1-NA and 2-NA. In this case, the FDA-hydrolyzing enzymes are 9

1

more labile than the 1-NA and 2-NA hydrolyzing esterases. The other possibility is that FDA is a

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more sensitive substrate for esterases than 1-NA and 2-NA. Further detailed research would be

3

required for the identification of the enzymes that catalyze FDA hydrolysis. Taking all of these data

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together, the in vivo FDA hydrolysis assay provides a sensitive evaluation of cellular activity. When

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this assay is applied to other plant species, optimization of the measurement protocol for in vivo

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FDA hydrolysis may be required for cultured cells that produce endogenous fluorescent or colored

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substances, such as chlorophyll and carotene, etc.

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In vitro cultured cells rapidly respond to physical conditions (e.g., temperature or light) or to

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physiologically active substances such as phytohormones, toxins, and chemical components of the

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medium. Cell viability is a basic parameter for the evaluation of cellular responses to changes in the

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culture conditions. When the growth of cultured plant cells is very slow, as was the case for S. alba

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cells, the determination of the effects of changes in culture conditions on cell growth usually

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requires an extended period of time. The in vivo FDA hydrolysis assay can be applied to any type of

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plant suspension culture or to callus cultures and is useful for acceleration of research progress, if

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the amount of cells is sufficient for the FDA hydrolysis assay.

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Acknowledgements

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We thank Dr. Daisuke Umeno and Mr. Masahiro Tominaga for their technical assistance in

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fluorescence microscopy observations.

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References

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enzymatic hydrolysis of fluoresceinic esters. Proc. Natl. Acad. Sci. USA 55 (1966) 134-141.

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[4] T. Nishida, N. Ohnishi, H. Kodama, A. Komamine, Establishment of synchrony by starvation

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and readdition of auxin in suspension cultures of Catharanthus roseus cells. Plant Cell Tiss. Organ Cult. 28 (1992) 37-43. [5] A. Yano, K. Suzuki, H. Uchimiya, H. Shinshi, Induction of hypersensitive cell death by a fungal

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protein in cultures of tobacco cells. Mol. Plant-Microbe Interact. 11 (1998) 115-123.

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[6] J. Schnürer, T. Rosswall, Fluorescein diacetate hydrolysis as a measure of total microbial

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activity in soil and litter. Appl. Environ. Microbiol. 43 (1982) 1256-1261. [7] Y. Kawana, H. Sasamoto, Stimulation effects of salts on growth in suspension culture of a

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mangrove plant, Sonneratia alba, compared with another mangrove, Bruguiera sexangula and

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non-mangrove tobacco BY-2 cells. Plant Biotechnol. 25 (2008) 151-155.

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[8] A. Hasegawa, A. Kurita, S. Hayashi, T. Fukumoto, H. Sasamoto, Halophilic and salts tolerant

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protoplast cultures of mangrove plants, Sonneratia alba and Avicennia alba. Plant Biotechnol.

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Rep. (2013) doi: 10.1007/s11816-012-0251-2

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[9] Y. Kawana, R. Yamamoto, Y. Mochida, K. Suzuki, S. Baba, H. Sasamoto, Generation and

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maintenance of suspension cultures from cotyledons and their organogenic potential of two

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mangrove species, Sonneratia alba and S. caseolaris. Plant Biotechnol. Rep. 1 (2007) 219-226.

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[10] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue

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cultures. Physiol. Plant. 15 (1962) 473-49.

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[11] A. Burlina, L. Galzigna, A new and simple procedure for serum arylesterase. Clin. Chim. Acta 39 (1972) 255-257.

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[12] B. Balen, M. Krsnik-Rasol, I. Zadro, V. Simeon-Rudolf, Esterase activity and isoenzymes in

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relation to morphogenesis in Mammillaria gracillis Pfeiff. tissue culture. Acta Bot. Croat. 63

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(2004) 83-91.

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Figure legends

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Fig. 1. Growth of S. alba cells. (A) Growth curve. Cells were maintained in MS medium containing

4

50 mM NaCl. Changes in the SCV over the course of 21 d are shown. (B) Growth in the presence

5

of NaCl. S. alba cells were maintained in MS medium containing 0 mM, 50 mM or 100 mM NaCl.

6

Fresh weight was determined at 0 d (grey bar) and 21 d of subculture (white bar). (C) Vital staining

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of cells with FDA. Cells were incubated with FDA for 5 min. Upper panels are bright field pictures

8

and lower panels are fluorescence pictures. Scale bar indicates 100 µm.

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Fig. 2. Establishment of FDA hydrolysis assay. (A) FDA hydrolysis activity in the cell-free

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supernatant. S. alba cells were removed from the 21-d-old cultures by filtration, and FDA was

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added to the resultant filtrate solution. Two culture conditions were provided; one was free of NaCl,

13

and the other contained 50 mM NaCl. Fluorescein can be seen as a yellow pigment. Tube 1, fresh

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MS medium with FDA; tube 2, cell-free filtrate; tube 3, cell-free filtrate with acetone; tube 4, cell-

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free filtrate with FDA. All tubes were place at room temperature for 60 min. (B) Fluorescein

16

production with various amounts of FDA. Cells were collected from a culture containing 50 mM

17

NaCl. Hydrolysis was performed with 0.5 g of cells for 60 min. (C) Fluorescein production over

18

time. The 21-d-old cells were collected from the NaCl-free (closed triangle), 50 mM NaCl (open

19

circle) or 100 mM NaCl (closed circle) cultures, and 0.5 g of cells was subjected to FDA hydrolysis

20

for 60 min. (D) Fluorescein production by different amounts of S. alba cells. The symbols are the

21

same as those in Fig. 2C. S. alba cells were incubated with FDA for 60 min. All symbols represent

22

the means of four replicates. Bars indicate SD.

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Fig. 3. Effect of osmotic stress on S. alba cells. (A) Evaluation of effects of mannitol in 4-d-old

25

cells. Cells maintained in the NaCl-free MS medium (white bars) were transferred to fresh MS

26

medium with or without 100 mM mannitol. Similarly, cells grown in the MS medium with 50 mM 13

1

NaCl (black bars) were transferred to fresh MS medium supplemented with NaCl or mannitol. Cells

2

were collected 4 d after transfer. The growth of cells was determined by measurement of FW (upper

3

panel), and cellular metabolic activity was evaluated by in vivo FDA hydrolysis (lower panel). (B)

4

Effects of mannitol on cell growth after prolonged culture. Cells maintained in the 50 mM NaCl-

5

containing MS medium were transferred to fresh MS medium with or without 100 mM mannitol.

6

Cell growth was monitored by determination of SCV after 11 d of subculture (white bars). Grey

7

bars indicate SCV that was determined just after transfer to fresh medium. (C) Esterase activity in

8

the protein extracts. The enzyme activity was determined with 1-NA, 2-NA and FDA as substrates

9

in the 4-d-old cells grown with 100 mM mannitol or with 50 mM NaCl.

14

1

15

1

16

1

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