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Journal of Plant Physiology 171 (2014) 1385–1391

Contents lists available at ScienceDirect

Journal of Plant Physiology

journa l h om epage: www.elsev ier .com/ locate / jp lph

hysiology

ffect of NaCl on ionic content and distribution inuspension-cultured cells of the halophyte Sonneratia alba versus thelycophyte Oryza sativa

anabu Hayatsua,b,∗, Suechika Suzukia,b, Ai Hasegawac, Shinpei Tsuchiyac,amako Sasamotob,c,d

Department of Biological Sciences, Faculty of Science, Kanagawa University, Hiratsuka, Kanagawa 259-1293, JapanResearch Institute for Integrated Science, Kanagawa University, Hiratsuka, Kanagawa 259-1293, JapanGraduate School of Environment and Information Sciences, Yokohama National University, Yokohama 240-8501, JapanFaculty of Environment and Information Sciences, Yokohama National University, Yokohama 240-8501, Japan

r t i c l e i n f o

rticle history:eceived 21 January 2014eceived in revised form 23 June 2014ccepted 23 June 2014vailable online 1 July 2014

eywords:ryosectionsalophilic natureangrove plant

alt tolerance-ray microanalysis

s u m m a r y

The effect of a high concentration of NaCl on the intra- (cytoplasmic matrix and vacuole) and extracellular(cell wall) distribution of Na, Cl, K, Mg, Ca, S, and P was investigated in suspension-cultured cells of themangrove halophyte Sonneratia alba and compared to cultured cells of glycophytic rice (Oryza sativa). Nosignificant differences were observed in ultrastructural features of cluster cells of both species culturedwith and without 50 mM NaCl. Quantitative X-ray microanalysis of cryosections of the cells culturedin the presence of 50 mM NaCl showed that the Na concentration ([Na]) and Cl concentration ([Cl])significantly increased in all three cell components measured. In S. alba, the [Na] was highest in thevacuole and lowest in the cytoplasmic matrix, while the [Cl] was highest in the cell wall and lowest inthe cytoplasmic matrix. In O. sativa, however, the [Na] and [Cl] were highest in the cell wall, and the[Na] was lowest in the cytoplasmic matrix. Thus, the possible activities for Na and Cl transport from thecytoplasmic matrix into the vacuole were greater in S. alba than in O. sativa, suggesting that halophilicmangrove cells gain salt tolerance by transporting Na and Cl into their vacuoles. In O. sativa, the additionof NaCl to the culture medium caused no significant changes to the intracellular concentrations of variouselements, such as K, P, S, Ca, and Mg, which suggests the absence of a direct relationship with the transportNa and Cl. In contrast, a marked decrease in the Ca concentration ([Ca]) in the cytoplasmic matrix and

vacuole and an approximately two-fold increase in the P concentration ([P]) in the cytoplasmic matrixwere found in S. alba, suggesting that the decrease in the [Ca] is related to the halophilic nature of S.alba (as indicated by the inward movement of Na+ and Cl−). The possible roles of a Na+/Ca2+ exchangemechanism in halophilism and the effect of the [P] on the metabolic activity under saline conditions arediscussed.

© 2014 Elsevier GmbH. All rights reserved.

ntroduction

More than 100 species of different families of mangrove

lants grow in brackish water in tropical and subtropical areasTomlinson, 1986; Spalding et al., 2010). The salt tolerance mecha-isms of these species at the cellular level are of interest for future

Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; DW, dry weight; MS basaledium, Murashige and Skoog basal medium; PCV, packed cell volume.∗ Corresponding author. Tel.: +81 0463 59 4111x2808; fax: +81 0463 58 9684.

E-mail addresses: hayatsu-bio@kanagawa-u.ac.jp, hayatsu bio@yahoo.co.jpM. Hayatsu).

ttp://dx.doi.org/10.1016/j.jplph.2014.06.008176-1617/© 2014 Elsevier GmbH. All rights reserved.

use in biotechnology (Kawana et al., 2008). Because cultured cellsthat are derived from mangrove plants are considered useful for thestudy of tolerance mechanisms, suspension cultures of mangroveplants have been developed from three families: Avicenniaceae(Hayashi et al., 2009), Rhizophoraceae (Kura-Hotta et al., 2001),and Sonneratiaceae (Kawana et al., 2007; Yamamoto et al., 2011).The effects of adding high concentrations (10–200 mM) of the ionsNa+, K+, Mg2+, Ca2+, Cl−, and SO4

2−, which are contained in seawa-ter, on the growth of these cells were investigated and compared to

those of glycophytic plants (Kawana and Sasamoto, 2008; Hayashiet al., 2009). Lower concentrations of these ions, except for Na+, arepresent as major components of the basal media for plant tissueand cell cultures.

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Sonneratia alba is a mangrove plant that can grow in the mosteaward side of mangrove forests. A suspension culture of S.lba was induced from the cotyledons (Kawana et al., 2007) inurashige and Skoog (MS) basal medium (Murashige and Skoog,

962), which is commonly used in tissue cultures for not onlyerbaceous plants but also woody plants, and the effects of theonstituent ions of seawater at high concentrations on the growthf these cells were investigated. The growth was markedly stimu-ated with the addition of 50–100 mM NaCl. Although the effectiveoncentrations were different, the halophilic nature of S. albauspension-cultured cells to other ions, including K+ (10 mM), Mg2+

10–25 mM) and Ca2+ (10–25 mM), has also been reported (Kawanand Sasamoto, 2008). In addition, the cotyledon protoplasts of S.lba were halophilic to these ions, except for Ca2+ (Hasegawa et al.,013). Although tolerance to NaCl was observed in the suspension-ultured cells and protoplasts of other mangrove plants, suchs Avicennia alba (Hayashi et al., 2009) and Bruguiera sexangulaFukumoto et al., 2004; Kawana and Sasamoto, 2008), S. alba isnique because of its halophilic tolerance to NaCl.

To determine the cellular mechanisms underlying the halophilicature of mangrove plants, we studied the intra- (cytoplasmicatrix and vacuole) and extracellular (cell wall) distribution of var-

ous elements, Na, Cl, K, Mg, Ca, S, and P, using electron probe X-rayicroanalysis (Hayatsu et al., 2012) in S. alba suspension-cultured

ells that were treated with 50 mM NaCl. For comparison, we pre-ared the suspension-cultured cells of Oryza sativa using the sameS basal medium with and without 50 mM NaCl.

aterials and methods

uspension culture

Suspension cultures of Sonneratia alba J. Smith were developedrom cotyledons (Kawana et al., 2007) and sub-cultured at 2- to-week intervals. The culture medium was Murashige and SkoogMS) basal medium (Murashige and Skoog, 1962), including var-ous elements as the major salt components (6 mM Cl, 3 mM Ca,0.3 mM K, 1.5 mM Mg, 1.25 mM P, 60 mM N, and 1.73 mM S), Na

n trace amounts (0.2 mM), 0.1 �M 2,4-dichlorophenoxyacetic acid2,4-D), and 3% sucrose. To examine NaCl tolerance, the experimen-al medium was prepared by adding 50 mM NaCl to the culture

edium. The cells were cultured in 15–20 mL of medium using a00-mL flask at 30 ◦C on a rotary shaker at 100 rpm.

A suspension culture (Oc strain, RPC00031) of Oryza sativaas provided by RIKEN BRC (Bio-Resource Center) through theational Bio-Resource Project of MEXT (Ministry of Education,ulture, Sports, Science and Technology), Japan. The culture wasub-cultured in MS basal medium containing various elementss previously described, as well as 4.5 �M 2,4-D and 3% sucroset 10- to 14-d intervals in 20 mL of medium in a 100-mL flaskn a rotary shaker at 27 ◦C. To examine the NaCl tolerance, thexperimental medium was prepared by adding 50 mM NaCl tohe culture medium. The effect of different concentrations ofaCl (10–200 mM) was examined by the small-scale method (ss-ethod) (Kawana and Sasamoto, 2008). Using 24-well culture

lates, the packed cell volume (PCV) and dry weight (DW) of cellshat were centrifuged in a microtube (MCT-150-C, Axygen Inc.)ere measured.

onventional electron microscopy

Agarose-solidified cell clusters that were derived from the sus-ension cultures of S. alba and O. sativa were fixed with a 6%lutaraldehyde solution (pH 7.2 by 0.1 M phosphate buffer) andost-fixed with a 2% osmium tetraoxide solution at 4 ◦C overnight.

siology 171 (2014) 1385–1391

The fixed specimens were dehydrated with a graded series of ace-tone and embedded in resin Quetol 812 (Nisshin EM Co. Ltd., Tokyo,Japan). Ultrathin sections of ∼70 nm thickness were cut on anultramicrotome (Ultracut-N; Reichert-Jung, Vienna, Austria), andstained with uranyl acetate and lead citrate. The sections wereexamined with a JEM 1230 transmission electron microscope (JEOL,Akishima, Tokyo, Japan) operated at an accelerating voltage of100 kV.

Rapid freezing and cryosection preparation

Regarding electron microscopic observations, 10 mL of 1- to 2-week-old suspension-cultured cells of S. alba and O. sativa werecentrifuged in a 15-mL tube at 800 rpm for 5 min. Agarose (TypeVII, Sigma A-4018) was then dissolved (2%) with supernatantmedium at 60 ◦C for 20 min. The precipitate, which was composedof suspension-cultured cells, was mixed with the above agarosemedium at room temperature and solidified. The successive pro-cedures were the same as those described in a previous study(Hayatsu et al., 2012). Briefly, the samples were high-pressurefrozen (EM-PACT, Leica, Austria) and stored in liquid N2. Cryosec-tions (Cryoultramicrotome, Ultracut UCT/EM FCS, Leica, Austria)were freeze-dried (VDF300S, Vacuum Device, Inc., Mito, Japan)at 1.3 × 10−4 Pa and −80 ◦C for 6 h. The cryosections were lightlyevaporated in vacuo with carbon for quantitative X-ray microanal-ysis.

Electron probe X-ray microanalysis

Electron probe X-ray microanalysis was performed using ananalytical electron microscope (JEM 1230; JEOL, Akishima, Tokyo,Japan) that was equipped with an energy-dispersive X-ray micro-analyzer (MiniCup/EX-14033JTP; JEOL, Akishima, Tokyo, Japan).The inside of the electron microscope column was cooled usinga cold trap apparatus to avoid specimen contamination. Freeze-dried cryosections on the Ni grid were equipped on a Be-stageof the cryotransfer holder (G626DH; Gatan, Tokyo, Japan) andentered into the electron microscope. The cryosections were thencooled to −130 ◦C by filling the container of the cryotransfer holderwith liquid N2. In the X-ray microanalyses (spot analysis), theelectron microscope was operated at an accelerating voltage of80 kV, and the current density of the electron beam was main-tained at 75.0 pA/cm2 in a fixed area (diameter, 0.1 �m) under amagnification of ×15,000. X-ray emissions from the cryosectionwere collected for 200 s. We monitored the detection sensitiv-ity of the microanalyzer at a dead time of ∼20% and ∼2000 atcounts per second (cps) and confirmed the stability of detec-tion.

The elemental concentrations were calculated from X-ray spec-tra in mmol/kg DW based on Hall’s quantitative equation (Hall,1971; Shuman et al., 1976; Suzuki et al., 2004). X-ray emissionsof up to 10 keV were collected. X-ray counts of each spectralpeak (peak intensity P) and the integral X-ray count in the regionof 4.5–5.5 keV for a representative intensity of the X-ray contin-uum (background intensity B) were calculated (Suzuki et al., 2004;Hayatsu et al., 2012). The concentration of each element was cal-culated based on the P/B intensity ratio using the software NORANSystem SIX (Thermo Electron Co., Middleton, WI, USA). The experi-ments were repeated with independently prepared samples. Thedata were averaged from ten values for each element, and the

standard error was calculated. The Student’s t distribution was usedto determine the significance of the differences between the ele-mental concentrations of the cell clusters that were cultured withand without 50 mM NaCl.

M. Hayatsu et al. / Journal of Plant Phy

Fig. 1. Effects of the addition of NaCl concentrations on the growth of suspensioncultures from O. sativa. The ordinate gives relative values of the PCV and DW asmm

R

SN

aoutwt

a typical cluster cell of O. sativa that was cultured without 50 mM

F51

easured by the ss-method with a 24-well culture plate. The bar on each plottingark shows the S.E.M.

esults and discussion

alt tolerance of suspension-cultured cells of O. sativa with 50 mMaCl

As a comparison with the halophilic nature of S. alba (Kawanand Sasamoto, 2008), the effect of high concentrations of NaCln the growth of suspension cultures of O. sativa were examinedsing 24-well culture plates (the ss-method). As shown in Fig. 1,

he packed cell volume (PCV) was reduced by approximately 10%hen the NaCl concentration was increased to 50 mM and con-

inued to decrease as the NaCl concentration increased further.

ig. 2. Electron microscope images from the chemically fixed and resin-embedded cell clu0 mM NaCl (b). Examples of O. sativa cells cultured without (c) and with 50 mM NaCl (0 �m.

siology 171 (2014) 1385–1391 1387

Similar decreases were observed in the PCV of the suspension cul-ture of another glycophytic herbaceous plant, tobacco (Kawana andSasamoto, 2008). In this study, the dry weight (DW) was first mea-sured using the ss-method. The DW did not significantly decreaseat concentrations of up to 200 mM NaCl. The suspension-culturedcells of O. sativa were tolerant to 50 mM NaCl.

Ultrastructures of suspension cultures

The suspension cultures of S. alba and O. sativa, cultured withand without 50 mM NaCl, were observed by conventional elec-tron microscopy. In both of the species, the cluster cells that werederived from the suspension-cultured cells were 20–40 �m indiameter (Fig. 2). The cell walls were well developed between thecluster cells and completely surrounded the outermost cells of acluster. Fig. 2a shows an example of the cluster cells of S. alba whencultured without 50 mM NaCl. These cells generally contained anucleus, mitochondria, plastids, fragmented endoplasmic reticula,and a large central vacuole. Fig. 2b shows the cluster cells of S. albawhen cultured with 50 mM NaCl. No significant differences wereobserved in the ultrastructural features of the cluster cells of S. albathat were cultured with and without 50 mM NaCl. Fig. 2c shows

NaCl, which included a nucleus, mitochondria, plastids, and endo-plasmic reticula. In the section views, the vacuole appeared to bedivided into many sub-vacuoles. Fig. 2d shows a cluster cell of O.

sters of S. alba and O. sativa. Examples of S. alba cells cultured without (a) and withd). In both species, the cells forming clusters contained a large vacuole. Scale bars

1388 M. Hayatsu et al. / Journal of Plant Physiology 171 (2014) 1385–1391

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ig. 3. Cryosection images of parts of the cluster cells that were cultured with 50 ms a nucleus (N) and vacuoles (V). The central vacuoles contained many electron-de

ativa that was cultured with 50 mM NaCl. No significant differ-nces were observed in the structural features of the cluster cellsf O. sativa that were cultured with and without 50 mM NaCl.

ltrastructure of the frozen cells in cryosections

Fig. 3 shows an example of the electron micrographs of cryosec-ions that were cut from the frozen cell clusters of S. alba (Fig. 3a)nd O. sativa (Fig. 3b) that were cultured with 50 mM NaCl. Withoutny chemical fixation before rapid freezing, the cells retained theirltrastructures well. Organelles, such as nuclei and vacuoles, werelearly observed with cell walls in these cryosections. The centralacuoles contained many electron-dense particles. No structuralifferences were observed in the cryosections of the cluster cells ofoth species that were derived from cultures that were incubatedith MS medium with and without 50 mM NaCl.

lectron probe X-ray microanalysis

A spot analysis was carried out at the cell wall, the cytoplasmicatrix, and the vacuolar lumen in the cryosections that were cut

rom the frozen cell clusters of S. alba and O. sativa that were cul-ured with and without 50 mM NaCl. The results are summarized inable 1 for S. alba and Table 2 for O. sativa. The given elements werehose that were detected in X-ray spectra as a significant spectral

eak. The elemental concentrations are indicated in mmol/kg DW.oncerning the results of Student’s t distribution, the differencesetween the mean values with P < 0.01 were considered significantTables 1 and 2).

able 1he concentrations of various elements in the cell wall, cytoplasmic matrix and vacuole i

Cell wall Cytoplasmic matri

Element Without 50 mM NaCl With 50 mM NaCl Without 50 mM Na

Na 6.5 ± 1.7 168.6 ± 14.3**** 2.1 ± 0.9

Mg 20.8 ± 2.1 15.9 ± 1.9* 7.4 ± 1.6

P 4.6 ± 0.9 5.7 ± 1.8* 14.3 ± 2.7

S 12.6 ± 1.8 14.8 ± 2.3* 28.1 ± 8.1

Cl 7.6 ± 1.6 162.7 ± 19.3**** 4.0 ± 1.4

K 102.2 ± 7.1 126.5 ± 14.2* 43.2 ± 4.9

Ca 69.8 ± 7.3 41.8 ± 7.0*** 20.5 ± 4.4

he values are mmol/kg DW (mean ± S.E.M., N = 10). The significances of the differences frlso shown.

* P > 0.1.** P < 0.1.

*** P < 0.05.**** P < 0.01.

Cl. The parts of the cluster cells of S. alba (a) and O. sativa (b) have organelles, sucharticles. Scale bars 5 �m.

In the cluster cells of S. alba, the mean value of the Na concen-tration ([Na]) was 6.5 mmol/kg DW in the cell wall, 2.1 mmol/kgDW in the cytoplasmic matrix, and 5.5 mmol/kg DW in thevacuolar lumen. When the cluster cells were incubated in themedium containing 50 mM NaCl, the [Na] markedly increasedin the cell wall (168.6 mmol/kg DW, ∼26-fold), the cytoplasmicmatrix (46.7 mmol/kg DW, ∼22-fold), and the vacuolar lumen(224.6 mmol/kg DW, ∼41-fold). The [Na] among the three cell com-ponents was highest in the vacuole and lowest in the cytoplasmicmatrix. The significant changes in the [Na] are schematically shownin Fig. 4, with concentration changes in Cl and Ca in S. alba and O.sativa.

Similar changes were observed in the Cl concentration ([Cl])in S. alba. Without 50 mM NaCl in the cultured medium, the[Cl] was similar to the [Na] in the cell wall and cytoplasmicmatrix but was slightly higher than the [Na] in the vacuolarlumen. When the cluster cells were cultured in the mediumwith 50 mM NaCl, the [Cl] markedly increased in every cellcomponents. The [Cl] was similar to the [Na] in the cell wall andcytoplasmic matrix, but was lower than the [Na] in the vacuolarlumen. As was observed for the [Na] in both cluster cells culturedwith and without 50 mM NaCl, the [Cl] was lowest in the cyto-plasmic matrix among the three cell components. These resultssuggested that Na and Cl accumulated in the vacuole to maintain alow [Na] and [Cl] in the cytoplasmic matrix.

In the cluster cells of O. sativa that were cultured without 50 mMNaCl, the mean value of [Na] was 5.4 mmol/kg DW in the cellwall, 1.0 mmol/kg DW in the cytoplasmic matrix and 1.5 mmol/kgDW in the vacuolar lumen. When 50 mM NaCl was added to the

n suspension-cultured cells of S. alba.

x Vacuole

Cl With 50 mM NaCl Without 50 mM NaCl With 50 mM NaCl

46.7 ± 12.8**** 5.5 ± 1.9 224.6 ± 32.6****

7.9 ± 1.4* 17.4 ± 3.4 11.0 ± 2.9*

26.8 ± 3.4**** 9.0 ± 2.8 7.9 ± 2.1*

15.5 ± 2.9** 18.4 ± 2.0 49.1 ± 11.8***

53.2 ± 7.9**** 20.2 ± 4.1 118.0 ± 18.1****

51.9 ± 7.7* 175.6 ± 23.1 277.6 ± 34.9***

5.0 ± 1.9**** 33.3 ± 4.3 1.7 ± 1.0****

om the values of cell clusters that were cultured with and without 50 mM NaCl are

M. Hayatsu et al. / Journal of Plant Physiology 171 (2014) 1385–1391 1389

Table 2The concentrations of various elements in the cell wall, cytoplasmic matrix and vacuole in suspension-cultured cells of O. sativa.

Cell wall Cytoplasmic matrix Vacuole

Element Without 50 mM NaCl With 50 mM NaCl Without 50 mM NaCl With 50 mM NaCl Without 50 mM NaCl With 50 mM NaCl

Na 5.4 ± 2.2 232.8 ± 19.3**** 1.0 ± 0.6 89.6 ± 13.2**** 1.5 ± 1.1 108.5 ± 20.6****

Mg 22.0 ± 5.7 10.7 ± 2.8* 14.8 ± 3.9 3.6 ± 1.7*** 20.6 ± 4.7 7.8 ± 2.5***

P 9.0 ± 2.1 17. 2 ± 1.0**** 43.4 ± 9.8 27.7 ± 5.6* 15.2 ± 5.8 5.2 ± 1.8*

S 17.4 ± 2.2 16.8 ± 2.8* 23.7 ± 2.2 21.1 ± 2.2* 27.0 ± 8.4 13.6 ± 2.9*

Cl 25.0 ± 3.3 142.0 ± 14.5**** 17.0 ± 4.4 67.6 ± 13.6*** 11.0 ± 2.5 38.3 ± 7.1***

K 145.5 ± 37.6 202.1 ± 12.8* 35.2 ± 3.0 62.1 ± 6.9*** 40.5 ± 9.2 61.4 ± 8.3*

Ca 14.4 ± 3.5 10.1 ± 2.8* 11.9 ± 1.3 8.6 ± 1.6* 8.2 ± 1.9 10.3 ± 1.8*

The values are mmol/kg DW (mean ± S.E.M., N = 10). The significances of the differences from the values of cell clusters that were cultured with and without 50 mM NaCl arealso shown.

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ulture medium, the [Na] markedly increased in every cell com-onent, similar to S. alba. Increases were detected in the [Na] inhe cell wall (232.8 mmol/kg DW, ∼43-fold), in the cytoplasmic

atrix (89.6 mmol/kg DW, ∼90-fold), and in the vacuolar lumen108.5 mmol/kg DW, ∼72-fold). The [Na] was highest in the cellall and lowest in the cytoplasmic matrix. The [Na] in the vac-ole of O. sativa cells was not high, which was in contrast to thatbserved in the S. alba cells.

In the cluster cells of O. sativa, the [Cl] increased in the threeell components when the cells were cultured in the medium with0 mM NaCl. However, the increase in the [Cl] in the vacuolar lumenas not as marked as that of the [Na]. The [Cl] was lowest in the vac-olar lumen. These results indicate that the mechanism underlyingl transport functions independent of that of Na transport.

ossible activities for Na and Cl transport

To elucidate the mechanism of transport in suspension-culturedells, it is reasonable to compare the [Na] and [Cl] in mmol/kg DWTable 1, Fig. 4). Furthermore, the possible activity for Na trans-ort was estimated as the percentage increase from the difference

n [Na] or [Cl] between the cell components. The possible activ-

ty for Na transport in S. alba that was cultured with 50 mM NaClrom the cell wall to the cytoplasmic matrix was estimated at ∼28%,hile that from the cytoplasmic matrix to the vacuole was ∼481%.

he possible activity for Cl transport in S. alba that was cultured

ig. 4. Diagram of the cluster cells of S. alba (a) and O. sativa (b). The values indicate si0 mM NaCl to MS basal medium. The red, blue, and yellow arrows indicate increases, de%) surrounded by rectangles in the field of the cytoplasmic matrix are relative values to

y rectangles in the field of the vacuole are relative values to the concentration indicateacuole. (For interpretation of the references to color in figure legend, the reader is referr

with 50 mM NaCl from the cell wall to the cytoplasmic matrix wasestimated at ∼33%, while that from the cytoplasmic matrix to thevacuole was ∼222%.

However, the possible activity for Na transport in O. sativa thatwas cultured with 50 mM NaCl from the cell wall to the cytoplasmicmatrix was estimated at ∼38%, while that from the cytoplasmicmatrix to the vacuole was ∼121%. In contrast, the possible activityfor Cl transport in O. sativa that was cultured with 50 mM NaCl fromthe cell wall to the cytoplasmic matrix was estimated at ∼48%. Theactivity for Cl transport from the cytoplasmic matrix to the vacuolewas ∼57%, which was markedly lower than the values for Na.

The possible activities for Na and Cl transport from the cyto-plasmic matrix to the vacuole were estimated to be very high inS. alba, indicating the positive accumulation of these elements fora halophilic nature. However, these activities were not so high forNa and Cl in O. sativa, although the change in their concentrationssuggested the possible existence of a salt tolerance mechanism. Ahigh rate of Na and Cl influx into the vacuolar lumen has also beenreported in isolated vacuoles of Atriplex leaf mesophyll cells (Matohet al., 1987). The Na+/H+ antiport mechanism (Blumwald and Poole,1985; Barkla et al., 1995; Oh et al., 2009) and the Cl− transportmechanism (Matile, 1978) in some halophyte plants work not only

salt tolerance. As well as these halophyte plants, these results sug-gest that the vacuoles in both S. alba and O. sativa that possess theseion transfer mechanisms play a significant role in maintaining a

gnificant changes in the [Na], [Cl], and [Ca] in mmol/kg DW with the addition ofcreases, and no significant changes in the concentrations, respectively. The valuesthe concentration indicated in the field of the cell wall. The values (%) surroundedd in the field of the cytoplasmic matrix. CW, cell wall; CM, cytoplasmic matrix; V,ed to the web version of the article.)

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390 M. Hayatsu et al. / Journal of Pla

onstant physiological concentration in the cytoplasmic matrix viahe uptake and/or accumulation of excess Na and Cl, which areuccessively increased by an influx from the cell wall componentextracellular space). The ability of vacuoles to regulate the [Na] inhe cytoplasmic matrix is very strong in S. alba and relatively lown O. sativa but is exactly the same in their tonoplasts.

oncentration changes in Ca and P

Tables 1 and 2 also show the concentrations of five elements,, P, S, Ca, and Mg, in addition to Na and Cl. These fundamentallements were included in the MS basal medium and are generallyound in plant tissues and cells. When the suspension-cultured cellsf O. sativa were incubated in the medium with 50 mM NaCl, no sig-ificant changes occurred in the concentrations of these elements

n the intracellular components, which suggested the absence of aorrelative transport mechanism for K, P, S, Ca, and Mg with Na andl under physiological conditions for salt tolerance.

A marked decrease in the Ca concentration ([Ca]) in the cytoplas-ic matrix and the vacuole and an approximately two-fold increase

n the P concentration ([P]) in the cytoplasmic matrix were found in. alba. [Ca] was markedly high (∼70 mmol/kg DW) in the cell wall of. alba cell clusters that were cultured without NaCl (Table 1, Fig. 4).his result indicates that the cell cluster in S. alba could sequesterr bind Ca2+ efficiently at the cell walls; the cell wall componentectin binds Ca2+ to form pectin gels (Jarvis, 1984). Meanwhile, theCa] detected in the cell wall was decreased to ∼42 mmol/kg DWhen the cultured cells were incubated with 50 mM NaCl. The sig-ificant decrease in the [Ca] upon incubation with 50 mM NaCl waslso confirmed in the cytoplasmic matrix and the vacuolar lumenn S. alba. This change in the [Ca] relative to [Na] and [Cl] suggestshe existence of some transport mechanisms. One of the potential

echanisms for this transport is the Na+/Ca2+ exchange mechanismhat has been detected in smooth and/or cardiac muscles (Reutert al., 1973; Suzuki and Sugi, 1989; Blaustein and Lederer, 1999)nd mainly extrudes excess Ca2+ in the cytoplasm together withctive Ca2+ pumps localized in the plasma membrane. The exist-nce of a Na+/Ca2+ exchanger-like protein related to salt toleranceas recently been reported in the plasma membrane of Arabidopsisells and is considered a bidirectional Ca2+ transporter that worksonversely to the movement of Na+ (Wang et al., 2012). In thease of S. alba, the extrusion of Ca may also occur from the vac-ole to the cytoplasmic matrix because the [Ca] decreases at the

nnermost vacuole. The activation of the Na+/Ca2+ exchange mech-nism in S. alba may be very important for avoiding the cytotoxicamage caused by an increase in the intracellular free Ca2+ con-entrations, and the observed [Ca] change may reflect a secondaryesponse to ensure NaCl tolerance. Meanwhile, no marked changesere observed in the [Ca] in O. sativa. Because the [Ca] was not soigh in the three cell components of cultured cells in O. sativa, inontrast to that observed in S. alba, the converse transport of Caelative to Na and Cl was not required.

A significant intracellular increase in the [P] was only found inhe cytoplasmic matrix of S. alba when cultured in the MS mediumith 50 mM NaCl. We cannot currently explain the reason for the

ncrease in the [P] but speculate that it may reflect an increasen the metabolic activity during cell growth induced by halophilicolerance. Further investigations are required to clarify this issue.

alt tolerance of mangrove plants

The intracellular distribution of ions was previously inves-

igated using protoplast and vacuole preparations from theuspension cells of B. sexangula (Kura-Hotta et al., 2001). We alsoeported the halophilic nature of protoplast cultures from theotyledons of S. alba and the salt tolerance of protoplasts from the

siology 171 (2014) 1385–1391

suspension cells of A. alba (Hasegawa et al., 2013). Protoplast isolat-ions from the suspension cells of S. alba (Kawana et al., 2009) and A.alba (Hasegawa et al., 2011) require strong enzymatic conditions,a long incubation time, and purification on a sucrose density gra-dient. Furthermore, as the cell wall is removed from protoplasts,the cells are exposed to osmotically stressed conditions. However,the method that was used in this study may have reduced possiblechanges in the distribution of ions that may have occurred dur-ing the isolation of protoplasts because of the rapid freezing of thecells. By comparing the quantitative X-ray microanalysis data ofthe suspension-cultured cells and their protoplasts, it will becomepossible to clarify the exact site of the regulation of the tolerance orhalophilic nature and osmotic tolerance to various ions in seawater.We may then be able to induce calluses or succeed in generating aprotoplast culture from recalcitrant mangrove species by manipu-lating the salt composition of the culture medium.

References

Barkla BJ, Zingarelli L, Blumwald E, Smith J. Tonoplast Na+/H+ antiport activity andits energization by the vacuolar H+-ATPase in the halophytic plant Mesembryan-themum crystallinum L. Plant Physiol 1995;109:549–56.

Blaustein MP, Lederer WJ. Sodium/calcium exchange: its physiological implications.Physiol Rev 1999;79:763–854.

Blumwald E, Poole RJ. Na/H antiport in isolated tonoplast vesicles from storage tissueof Beta vulgaris. Plant Physiol 1985;78:163–7.

Fukumoto T, Nakamura T, Suzuki M, Ogita S, Mimura T, Sasamoto H. Different effectsof four salts and pHs on protoplast cultures of a mangrove, Bruguiera sexangulasuspension cells, Populus alba leaves and tobacco BY-2 cells. Plant Biotechnol2004;21:177–82.

Hall TA. The Microprobe assay of chemical elements. In: Oster G, editor. PhysicalTechniques in Biological Research. New York: Academic Press; 1971. p. 157–275.

Hasegawa A, Hayashi S, Kurita A, Kaai F, Kawana Y, Fukumoto T, et al. Stimulatoryand inhibitory effects of abscisic acid on cell growth in protoplast cultures andthe relation to its endogenous levels in Avicenniaceae mangrove cells. MangroveSci 2011;8:11–8.

Hasegawa A, Kurita A, Hayashi S, Fukumoto T, Sasamoto H. Halophilic and saltstolerant protoplast cultures of mangrove plants, Sonneratia alba and Avicenniaalba. Plant Biotechnol Rep 2013;7:205–9.

Hayashi S, Kuriyama S, Kawana Y, Hasegawa A, Kurita A, Minagawa R, et al. Sti-mulatory effects of sea salts on cell growth in liquid culture of Avicenniaceaemangrove. Plant Biotechnol 2009;26:561–4.

Hayatsu M, Ono M, Hamamoto C, Suzuki S. Cytochemical and electron probe X-ray microanalysis studies on the distribution change of intracellular calciumin columella cells of soybean roots under simulated microgravity. J ElectronMicrosc 2012;61:57–69.

Jarvis MC. Structure and properties of pectin gels in plant cell walls. Plant Cell Environ1984;7:153–64.

Kawana Y, Sasamoto H. Stimulation effects of salts on growth in suspension cul-ture of a mangrove plant, Sonneratia alba, compared with another mangrove,Bruguiera sexangula and non-mangrove tobacco BY-2 cells. Plant Biotechnol2008;25:151–5.

Kawana Y, Yamamoto R, Mochida Y, Suzuki K, Baba S, Sasamoto H. Generation andmaintenance of suspension cultures from cotyledons and their organogenicpotential of two mangrove species, Sonneratia alba and S. caseolaris. PlantBiotechnol Rep 2007;1:219–26.

Kawana Y, Sasamoto H, Ashihara H. Mechanism of salt tolerance in mangrove plants.Bull Soc Sea Water Sci Jpn 2008;62:207–14 (in Japanese with English abstract).

Kawana Y, Kaai F, Sasamoto H. Abscisic acid stimulates cell divisions in cultures ofprotoplasts isolated from cotyledons and suspension cells of a mangrove plant,Sonneratia alba: small-scale measurements of abscisic acid and gibberellins inprotoplasts. Mangrove Sci 2009;6:9–15.

Kura-Hotta M, Mimura M, Tsujimura T, Washitani-Nemoto S, Mimura T. Highsalt-treatment-induced Na+ extrusion and low salt-treatment-induced Na+

accumulation in suspension-cultured cells of the mangrove plant Bruguierasexangula. Plant Cell Environ 2001;24:1105–12.

Matile P. Biochemistry and function of vacuoles. Annu Rev Plant Physiol1978;29:193–213.

Matoh T, Watanabe J, Takahashi E. Sodium, potassium, chloride, and betaine con-centrations in isolated vacuoles from salt-grown Atriplex gmelini leaves. PlantPhysiol 1987;84:173–7.

Murashige T, Skoog F. A revised medium for rapid growth and bioassay with tobaccotissue cultures. Physiol Plant 1962;15:473–97.

Oh DH, Leidi E, Zhang Q, Hwang SM, Li Y, Quintero FJ, et al. Loss of halophytism by

interference with SOS1 expression. Plant Physiol 2009;151:210–22.

Reuter H, Blaustein MP, Haeusler G. Na–Ca exchange and tension development inarterial smooth muscle. Philos Trans R Soc Lond B Biol Sci 1973;265:87–94.

Spalding MD, Kainuma M, Collins L. World atlas of mangroves. London: EarthscanPublications Ltd; 2010.

nt Phy

S

S

S

tein (AtNCL) involved in salt stress in Arabidopsis. J Biol Chem 2012;287:

M. Hayatsu et al. / Journal of Pla

human H, Somlyo AV, Somlyo AP. Quantitative electron probe microanalysis ofbiological thin sections: Methods and validity. Ultramicroscopy 1976;1:317–39.

uzuki S, Sugi H. Evaluation of the pyroantimonate method for detecting intracellu-

lar calcium localization in smooth muscle fibers by the X-ray microanalysis ofcryosections. Histochemistry 1989;92:95–101.

uzuki S, Hino N, Sugi H. Intracellular calcium translocation during thecontraction-relaxation cycle in scorpionfish swimbladder muscle. J Exp Biol2004;207:1093–9.

siology 171 (2014) 1385–1391 1391

Tomlinson PB. The botany of mangroves. London: Cambridge University Press; 1986.Wang P, Li Z, Wei J, Zhao Z, Sun D, Cui S. A Na+/Ca2+ exchanger-like pro-

44062–70.Yamamoto R, KawanaY. Minagawa R, Sasamoto H. Effects of sea salts on induction

of cell proliferation in liquid cultures of mangrove plants, Sonneratia caseolarisand S. alba. Am J Plant Sci 2011;2:35–42.