Plant Cell Physiol-2014-Chen-634-44

Ca 2+Signal Transduction Related to Neutral Lipid Synthesis in an Oil-Producing Green Alga Chlorella sp.C2

Hui Chen 1,Yunming Zhang 1,2,Chenliu He 1and Qiang Wang 1,*

1Key Laboratory of Algal Biology,Institute of Hydrobiology,Chinese Academy of Sciences,Wuhan 430072,Hubei,China 2

University of Chinese Academy of Sciences,Beijing 100094,China

*Corresponding author:E-mail,wangqiang@https://www.360docs.net/doc/fc5787783.html,;Fax,+86-27-68780123(Received November 12,2013;Accepted January 14,2014)

Changes in the cytosolic Ca 2+levels and the role of Ca 2+signal transduction in neutral lipid synthesis in Chlorella sp.C2under nitrogen starvation conditions were investigated.The results detected by using the scanning ion-selective elec-trode technique demonstrate that nitrogen starvation induced signi?cant Ca 2+in?ux across the plasma membrane into cells.Ca 2+?uorescence imaging and ?ow cytometry were used to estimate the effect of this Ca 2+in?ux on the generation of the Ca 2+signal,and the results showed that the cytosolic Ca 2+concentration increased transiently and then remained at a stable,high level when the cells were exposed to nitrogen starvation.However,the increase could be inhibited by pre-treatment with the Ca 2+channel block-ers ruthenium red,verapamil and GdCl 3,indicating that both the in?ux of Ca 2+from the extracellular space via Ca 2+channels that are localized in the plasma membrane and the release of Ca 2+from intracellular calcium storage via the internal calcium store were required for the generation and transduction of the Ca 2+signal.During nitrogen starva-tion,neutral lipid synthesis in Chlorella sp.C2in response to stress conditions was also inhibited to differing degrees by pre-treatment with the three Ca 2+channel blockers,demonstrating the regulation of Ca 2+via these Ca 2+chan-nels in neutral lipid synthesis.The results suggested that by transduction of extracellular stress signals into the cell and the regulation of the Ca 2+signal in neutral lipid synthesis,Ca 2+signal transduction played important roles in the re-sponse mechanism of Chlorella sp.C2to nitrogen starvation.Keywords:Ca 2+channel Ca 2+signal transduction Chlorella sp.C2 Neutral lipid synthesis Nitrogen starvation.

Abbreviations:CaM,calmodulin;CLSM,confocal laser scan-ning microscopy;Cs,control stage;FCM,?ow cytometry;LDFs,late oil droplet formation stage;N,nitrogen;N +,N-suf?cient;N–,N-de?cient;OD,oil droplet;ODFs,oil drop-let formation stage;PDFs,pre-oil droplet formation stage;PM,plasma membrane;RR,ruthenium red;SIET,scanning ion-selective electrode technique;TAG,triacylglycerol;TLC,thin-layer chromatography;VP,verapamil.

Introduction

Many plants are adversely affected by several environmental factors,including light,temperature,CO 2,O 2,water,nutrients,and stresses such as drought,low pH,salt and pathogen or predator attacks,that negatively affect their survival and devel-opment (Plieth 2001).Ca 2+is a ubiquitous intracellular second messenger in the signal transduction of environmental stimuli in plants (Sun et al.2006).When plants are forced to respond to environmental stimuli,the Ca 2+level rises rapidly and transi-ently in the cytoplasm as a result of either Ca 2+uptake from the extracellular space through the plasma membrane (PM)chan-nels or Ca 2+release from internal stores,such as the endoplas-mic reticulum or vacuoles (Sun et al.2006).The free Ca 2+molecules and the proteins that bind them are important con-served components of intracellular signaling networks (Bothwell and Ng 2005).Typical proteins that bind Ca 2+include calmodulin (CaM)and Ca 2+-or CaM-dependent enzymes [e.g.CaM domain protein kinases (CDPKs)and calcineurin],which translate the changes in the Ca 2+levels into the regulation of proteins in order to produce the appropriate response (Harmon et al.2000,Kim et al.2000,Plieth 2001,Nakamura et al.2006).Whether conditions are favorable or adverse,the intricate metabolic pathways of a cell are heavily in?uenced by its en-vironment.In the case of microalgae,specialized environmental factors can stimulate changes in metabolism (Rosenberg et al.2008).Among nutrient factors,nitrogen (N)limitation or N starvation is considered one main factor in?uencing cell growth and metabolism,and it is also an ef?cient environmen-tal pressure that is used to increase the lipid accumulation in microalgae.The general principle is that when there is insuf?-cient N for the protein synthesis that is required for growth,excess carbon from photosynthesis is channeled into storage molecules such as triglyceride or starch (Scott et al.2010).During N starvation,reduced photosynthetic rate,respiration rate and photochemistry ef?ciency also occurred,all of which resulted in the adverse effect on cell survival and growth (Zhang et al.2013).To respond to the adverse environment,the alga will be inclined to accumulate and store high energy com-pounds,such as lipids and starch (Mata et al.2010),in cells for recovery and re-growth under the appropriate conditions.

Plant Cell Physiol.55(3):634–644(2014)doi:10.1093/pcp/pcu015,available online at https://www.360docs.net/doc/fc5787783.html, !The Author 2014.Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.All rights reserved.For permissions,please email:journals.permissions@https://www.360docs.net/doc/fc5787783.html,

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The lipid content in Chlorella can be improved signi?cantly under N depletion conditions,and a linear relationship be-tween the N source concentration and the lipid content has been observed(Hsieh and Wu2009,Zhang et al.2013).In add-ition to the increase in the total lipid content in microalgal cells as a result of cultivation in N-depleted media,changing from normal media to N-depleted media will gradually change the lipid composition from free fatty acid-rich lipids to mostly tri-glyceride-containing lipids(Takagi et al.2000).Therefore,N limitation or N starvation could increase both the lipid and triglyceride contents in microalgal cells.

The green microalga Chlorella(Chlorophyta),which consists of approximately10species that can grow photoautotrophi-cally,mixotrophically and heterotrophically with a high bio-mass concentration,appears to be a particularly good option for biodiesel production(Petkov and Garcia2007).The oil con-tent in some species of Chlorella varies from approximately14% to63%of the dry weight,and the fatty acid composition ranges from C-14:0to C-20:0(Gouveia and Oliveira2009).

In recent years,the scanning ion-selective electrode tech-nique(SIET)has been reported to be a non-invasive method to obtain information about Ca2+as well as?uxes of other ions/ molecules across trans-membranes(Sun et al.2007).Although some information about Ca2+distributions and movements could be obtained by patch-clamp and?uorescence imaging methods(Chen et al.2011,Choi et al.2011,Maxwell and Blatter 2012,Lu et al.2013),SIET has become a novel and complemen-tary tool to the above techniques with its unique spatial and temporal resolutions,and was also an indispensable tool for identifying or verifying some functions of Ca2+?ux across the PM on signal transduction.

To obtain direct evidence of the involvement of Ca2+in the regulation mechanism of N starvation signaling in lipid accu-mulation in microalgae,in the present research the Ca2+?ux across the PM in Chlorella sp.C2—an oil-producing microalga that was isolated from the wild and identi?ed by its morph-ology and18S rRNA(unpublished data)—under N starvation was detected by using SIET.The change in the cytosolic Ca2+ levels and the role of Ca2+in neutral lipid accumulation under N starvation in Chlorella sp.C2were also investigated.The aim of this study was to investigate the role of Ca2+using Ca2+ channel blockers to elucidate the N starvation signal transduc-tion pathway in Chlorella sp.C2.

Results

Ca2+?ux in Chlorella sp.C2under N–treatment To determine whether Ca2+mediates the signal transduction under N-de?cent(N–)treatment to regulate lipid biosynthesis for stress adaptation,the N–treatment-induced Ca2+?ux in Chlorella sp.C2was measured.Fig.1shows that the Ca2+ef?ux across the PM could be observed in Chlorella sp.C2cells that were cultured in N-suf?cient(N+)BG11medium(Control). After the N–treatment,the Chlorella sp.C2cells exhibited marked decreases in the Ca2+ef?ux,which even changed to

a signi?cant Ca2+in?ux across the PM8d after the N–treat-

ment(Fig.1).This result indicates that the PM Ca2+channel

activity was enhanced by the N–treatment and that numerous

Ca2+molecules?ooded into cells using these Ca2+channels.

The increased Ca2+in?ux might form a Ca2+signal in Chlorella

sp.C2cells,regulating the cellular response mechanism to N

starvation via Ca2+-mediated signal transduction pathways.

Cytosolic Ca2+levels in Chlorella sp.C2cells that

were induced by the N–treatment

To detect the cytosolic Ca2+level changes in Chlorella sp.C2

after the N–treatment,a Ca2+-sensitive?uorescent dye,Fluo-3

AM,was used to monitor the cytosolic Ca2+in cells that were

cultured in N+medium and subjected to N starvation at four

key stages of neutral lipid accumulation.The?uorescence de-

tection in single cells using the Ratio Fluorescence Imaging

System showed that the N starvation caused a signi?cant in-

crease in the?uorescence intensity compared with the cells

that were cultured in the N+medium during all four of the

key stages of neutral lipid accumulation(Fig.2),suggesting that

the cytosolic Ca2+level in Chlorella sp.C2increased in order to

produce a Ca2+signal in response to N starvation.

To characterize further the cytosolic Ca2+level changes

during N starvation,large numbers of Chlorella sp.C2cells

(>104)during the four stages were analyzed using?ow cyto-

metry(FCM).In accordance with the results of the cytosolic

Ca2+level change in a single cell(Fig.2),a signi?cant increase in

the?uorescent intensity of Fluo-3AM using FCM was also

observed compared with the cells that were cultured under

normal N+growth conditions during all four stages(Fig.3). Moreover,FCM appeared to be more sensitive and statistical,

and the?uorescent signal in more than10000cells could be detected;an instantaneous increase in the?uorescent intensity,

which was undetected in a single cell(Fig.2a),could be de-

tected after several seconds of the N–treatment(Fig.3a).The

results demonstrate that the cytosolic Ca2+content increased transiently after the N–treatment and then maintained a

stable,high level during N starvation,indicating that the gen-

eration of the Ca2+signal was an instantaneous process fol-

lowed by the continuous activation of the Ca2+signal-mediated

signal transduction pathways to regulate the response process

of Chlorella sp.C2to N starvation.

The cytosolic Ca2+level increase derived from

both the extracellular environment and the

intracellular calcium stores

Normally,the rapid and transient increase of the cytosolic Ca2+

levels in plants that were subjected to environmental stimuli is

due to either Ca2+uptake from the extracellular space through

the PM channels or Ca2+release from the internal calcium

stores(Sun et al.2006).To determine the Ca2+source that

resulted in the increase of the cytosolic Ca2+levels in

Chlorella sp.C2under N starvation,an intracellular calcium

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store Ca 2+channel blocker [ruthenium red (RR)]and two PM Ca 2+channel blockers [verapamil (VP)and GdCl 3]were used.Fig.2shows the effect of the Ca 2+channel blockers on the cytosolic Ca 2+levels in single cells under N starvation.After the N–treatment,the cells that were pre-treated with RR,VP or GdCl 3exhibited lower cytosolic Ca 2+levels at all four stages compared with the cells that were not treated with a Ca 2+channel blocker (Fig.2),indicating that the N starvation-induced cytosolic Ca 2+level increase was blocked by RR,VP or GdCl 3and that either the Ca 2+channels in the PM or the internal calcium stores regulated the cytosolic Ca 2+https://www.360docs.net/doc/fc5787783.html,ing FCM,the blockage by RR,VP or GdCl 3of the increase in the cytosolic Ca 2+levels at all four stages in large numbers of cells under N starvation was also observed (Fig.3).These results from both the single cell and the multicellular level demon-strate that the increase in the cytosolic Ca 2+levels under N starvation was derived from both the extracellular environment and the intracellular calcium stores as a result of Ca 2+uptake via the PM Ca 2+channels and the Ca 2+release via the internal calcium stores.In addition,the cytosolic Ca 2+levels in Chlorella sp.C2cells that were pre-treated with RR were always less than those of the cells that were treated with VP or GdCl 3(Figs.2,3),suggesting the more marked role of the internal calcium stores in the regulation of the cytosolic Ca 2+levels for Ca 2+signal transduction.

In Figs.2and 3,the cytosolic Ca 2+levels in Chlorella sp.C2cells that were pre-treated with ionomycin did not increase signi?cantly at any of the four stages compared with the cells that were cultured in the N–medium.A tough cell wall is typical of some microalgae (e.g.Chlorella )(Running et al.1994,Ueno 2009).Therefore,it has been suggested that a small amount of ionomycin could permeate the cell wall and act as a Ca 2+ionophore in shuttling Ca 2+across the PM into the cytoplasm of Chlorella sp.C2.The increase in the cytosolic Ca 2+levels in the cells that were pre-treated with ionomycin might be independent of the Ca 2+ionophore but dependent on the Ca 2+channels,similar to the process in the cells that were cultured in the N–medium.

Ca 2+signal transduction-regulated neutral lipid synthesis during N starvation

In previous studies,four key stages of lipid accumulation,the control stage (Cs;0d),pre-oil droplet formation stage (PDFs;0–0.5d),oil droplet formation stage (ODFs;0.5–2d)and late oil droplet formation stage (LDFs;2–8d),were de?ned in Chlorella sorokiniana C3(Zhang et al.2013)and Chlorella sp.C2(unpub-lished data).To determine the regulation of Ca 2+signal trans-duction during neutral lipid accumulation and oil droplet (OD)formation in Chlorella sp.C2,the neutral lipid levels in the cells that were cultured under various conditions were examined at four stages of N starvation using thin-layer chromatography (TLC).During the ?rst two stages,no cells in any treatment accumulated detectable levels of neutral lipids (Fig.4a,b ).When the treatment was prolonged,the cells that were cul-tured in the N–medium began to accumulate neutral lipids at ODFs (Fig.4c ,lane 1),but the OD formation that was induced by the N starvation was blocked signi?cantly in the cells that were pre-treated with RR,VP or GdCl 3(Fig.4c ,lanes 2–4,Fig.4e ).In accordance with the cytosolic Ca 2+levels,although neutral lipid synthesis was beginning (Fig.4c ,lane 5,Fig.4e ),the cells that were pre-treated with ionomycin did not show a signi?cant increase in the neutral lipid content compared with the cells that were cultured in the N–medium (Fig.4c ,lane 1,Fig.4e ).Rising gradually with the duration of the stress,neutral lipids signi?cantly accumulated in the cells that were cultured in the N–medium at LDFs (Fig.4d ,lane 1),and the relative neutral lipid content reached 1.67times that of the reference substance (Fig.4f ).Similarly,the relative value of the neutral lipids in the cells that were pre-treated with ionomycin was 1.66times that of the reference substance (Fig.4f ).The neutral lipid accumulation in cells that were pre-treated with RR,VP or GdCl 3was also blocked to differing degrees,1.35,1.59or 1.55times that of the reference substance (Fig.4d ,lanes 2–4,Fig.4f ).All of these results indicate that the increase in the cytosolic Ca 2+level via these Ca 2+channels transmitted Ca 2+signals in order to regulate neutral lipid synthesis in Chlorella

sp.

Fig.1The total Ca 2+?ux rates over 5min in Chlorella sp.C2under N starvation.(a)Microphotographic examples of Ca 2+ion ?ux/voltage-clamp measurements.(b)Total ?ux rates of Ca 2+were detected at 0,0.5,2and 8d after N starvation.The columns represent the means of three replicated studies in each sample,with the SD of the means (t -test,P <0.05).The signi?cance of the differences between the control (0d)and other test values was tested using a one-way analysis of variance.*P <0.05vs.control.

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C2in response to N starvation,and ionomycin had no effect on neutral lipid synthesis.Moreover,the blocking effect of the three Ca 2+blockers on the neutral lipid accumulation at LDFs was obviously less than the effect on the OD formation at ODFs,indicating that the regulation of Ca 2+signal transduc-tion during neutral lipid synthesis occurred during the initial stage of OD formation and that the blockers deferred the OD formation that was induced by N starvation.

To visualize the regulation of OD formation by Ca 2+signal transduction,Chlorella sp.C2cells that were stained with BODIPY 505/515were observed using confocal laser scanning microscopy (CLSM).In agreement with the TLC results (Fig.4),no BODIPY 505/515?uorescence (green)was detected at the Cs (Fig.5,0d)or PDFs (Fig.5,0.5d)in any treatment.At the ODFs (Fig.5,2d),no ?uorescence was detected in the cells that were cultured in the N +medium,but a weak

green

Fig.2Effect of Ca 2+channel blockers under N starvation on the cytosolic Ca 2+levels in a single cell of Chlorella sp.C2.The ?uorescence of Fluo-3AM (green)was detected at 0d (a),0.5d (b),2d (c)and 8d (d)after N starvation;.(e)Fluorescence image of cells in each treatment at 0,0.5,2and 8d.N–,N starvation;RR,ruthenium red;VP,verapamil;Gd,GdCl 3;Ion,ionomycin;N +,N suf?cient.The data points and ?gures represent the means of three replicated studies in each sample.The size of the scale bar is shown directly on the image.

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?uorescence was detected in the cells that were cultured in the N–medium or pre-treated with ionomycin.However,the ?uor-escence intensity was weakened in the cells that were pre-trea-ted with RR,VP or GdCl 3.With prolonged N starvation,a strong green ?uorescence signal was observed at the LDFs in the cells that were cultured in the N–medium and that were pre-trea-ted with ionomycin (Fig.5,8d).The cells that were pre-treated with RR,VP or GdCl 3also exhibited a decrease in the ?uores-cence intensity.However,only a weak green ?uorescence was detected at LDFs in the cells that were cultured in the N +medium (Fig.5,8d).In addition,the Chl auto?uorescence (red)intensities in the cells in every treatment at each stage were nearly identical (Fig.2).

To characterize further the regulation of Ca 2+signal trans-duction during neutral lipid accumulation in response to N starvation,large numbers of Chlorella sp.C2cells (>104)at the four stages were analyzed using FCM.The ?uorescence intensity of BODIPY 505/515in the cell populations during N starvation increased with time,compared with that in the cells that were cultured in the N +medium,indicating a constant increase in the cellular neutral lipid content.The results from the FCM analysis also suggest that the Ca 2+ionophore iono-mycin did not play a role in the regulation of neutral lipid synthesis in Chlorella sp.C2(Fig.6).However,the constantly

increased ?uorescence intensity could be blocked in the cells that were pre-treated with RR,VP or GdCl 3during the OD formation (Fig.6),which corresponded to the ?ndings by CLSM (Fig.5)and TLC (Fig.4).

Total lipid contents in Chlorella sp.C2cells at ODFs and LDFs,the stages of OD preliminary formation and signi?cant accumulation,were also determined in order to evaluate dir-ectly the regulation of Ca 2+signal transduction.Similarly,the OD formation and accumulation in cells during N starvation could be blocked by pre-treatment with RR,VP or GdCl 3,and ionomycin did not have an effect (Fig.7),suggesting the regu-lation by Ca 2+signal transduction of lipid metabolism in Chlorella sp.C2under N starvation.All of the above results suggest that Chlorella sp.C2accumulated and stored lipids for recovery and re-growth under the appropriate conditions in the cells that were cultured under N starvation and that the N starvation response process was regulated by Ca 2+signal transduction pathways.

Discussion

As a key signal factor,speci?c changes in the cytosolic Ca 2+levels occur when plants or microalgae are exposed

to

Fig.3FCM analysis the cytosolic Ca 2+levels in Chlorella sp.C2under N starvation.The ?uorescence of Fluo-3AM was detected at 0d (a),0.5d (b),2d (c)and 8d (d)after N starvation.N–,N starvation;RR,ruthenium red;VP,verapamil;Gd,GdCl 3;Ion,ionomycin;N +,N suf?cient.The data points at each second represent the means of 2?103–3?103cells in three replicated studies with similar ?ndings.

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various environmental stresses,and the generation and trans-duction of a Ca 2+signal contribute to the transfer of extra-cellular stimuli to cells in order to regulate the response mechanism to environmental stresses (Chinnusamy et al.2004).The mechanisms giving rise to the changes in the cytosolic Ca 2+levels under environmental stresses are only beginning to be identi?ed in microalgae,and recent studies using Ca 2+chelators or Ca 2+channel blockers have impli-cated Ca 2+channels in the regulation of cytosolic Ca 2+.For example,a study by Torrecilla et al.(2001)demonstrated that both salinity and osmotic stress trigger transient in-creases in the intracellular free Ca 2+concentration in the cells of the cyanobacterium Anabaena sp.PCC7120,and both of the Ca 2+transients were completely blocked by the calcium chelator EGTA and were partially inhibited by the calcium channel blocker VP.In the present study,a transient increase in the cytosolic Ca 2+levels that was induced by N starvation and a stable,high level of cytosolic Ca 2+during N starvation were detected.The Ca 2+increase could also be markedly blocked by RR and partially inhibited by VP or GdCl 3(Figs.2,3),indicating that all of the corres-ponding Ca 2+channels,in particular the channels in the intracellular calcium stores,played special roles in the regu-lation of the cytosolic Ca 2+levels under N starvation.

To tolerate or adapt to these environmental stresses,micro-algae possess a variety of special response mechanisms,and some response mechanisms are regulated by the Ca 2+-mediated signal transduction pathways.In the halotolerant green alga Dunaliella bardawil ,the increase in the intracellular glycerol contents under hypertonic shock was sharply decreased by low concentrations of Ca 2+(1and 5mM)but increased by high concentrations of Ca 2+(10mM)(Issa 1996),which demonstrates that Ca 2+could regulate the intra-cellular glycerol content under osmotic stress.In a previous study,N starvation resulted in neutral lipid accumulation,and OD formation and signi?cant neutral lipid accumulation occurred after 2and 8d of starvation,respectively,in Chlorella sorokiniana C3(Zhang et al.2013)and Chlorella sp.C2(unpub-lished data).However,the Ca 2+signal transduction pathway in microalgae under nitrogen starvation conditions has not been studied,and the regulation mechanism of Ca 2+signal transduc-tion in lipid accumulation in microalgae is not yet known.In the present study,intracellular lipid especially neutral lipid synthe-sis during N starvation was blocked to differing degrees

by

Fig.4TLC analysis of the neutral lipid accumulation in Chlorella sp.C2cells under N starvation.The neutral lipid accumulation was detected at 0d (a),0.5d (b),2d (c)and 8d (d)after N starvation.The relative value of the neutral lipid content in each sample compared with the reference substance was calculated at 2d (e)and 8d (f).1/N–,N starvation;2/RR,ruthenium red;3/VP,verapamil;4/Gd,GdCl 3;5/Ion,ionomycin;6/N +,N suf?cient.An asterisk indicates the reference substance glyceryl trioleate.All of the ?gures for TLC are representative of three replicated studies with similar ?ndings.The columns represent the means of three replicated studies in each sample,with the SD of the means (t -test,P <0.01).The signi?cance of the differences between the control (N–)and test values was tested using a one-way analysis of variance.*P <0.05vs.control.

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pre-treatment with RR,VP or GdCl 3(Figs.4–7),indicating that Ca 2+signal transduction that is regulated by the corresponding Ca 2+channels signi?cantly contributed to the regulation of neutral lipid synthesis during N starvation.A similar ?nding has shown that the changes in the glycerol content and G3PDH activity in Dunaliella salina with respect to osmotic stress were partially inhibited by pre-treatment with the Ca 2+channel blockers LaCl 3,VP and RR,indicating that the in?ux of Ca 2+via Ca 2+channels is required for the transduction of the osmotic signal in order to regulate the osmotic responses of D.salina (Chen et al.2011).Therefore,Ca 2+channels are also involved in the Ca 2+signal transduction for the regulation of response mechanisms.

De Beer and Larkum (2001)found that the Ca 2+channel blockers VP and nifedipine had no effect on the Ca 2+dynamics or steady-state pro?les in the calcifying alga Halimeda discoidea .In the present study,the blocking effects of RR,VP and GdCl 3on the cytosolic Ca 2+levels (Figs.2,3)and neutral lipid

synthesis (Figs.4–6)were different,suggesting that the Ca 2+channel blockers acted on different channels or had different sites of action.This result also demonstrates various blocking effects and suggests an in?ux of Ca 2+from the extracellular space via the Ca 2+channels that are localized in the PM or from the intracellular calcium store via the Ca 2+channels that are localized in the calcium stores.In addition,no blocker could completely block the increase in the cytosolic Ca 2+levels (Figs.2,3)and neutral lipid synthesis (Figs.4–6)during N starvation.The tolerance and susceptibility to environmental stress are very complex phenomena,and Ca 2+signal transduc-tion is also a complex mechanism that might be regulated by the synergistic effect of the Ca 2+channels but not by the effect of any single channel.Furthermore,due to the diversity and complexity of microalgae,non-Ca 2+channel-regulated cyto-solic Ca 2+mechanisms might also exist.For example,Karimova et al.(2000)found that (i)the Ca 2+and Na +coun-ter-transporters span the membranes of two Dunaliella species (D.salina and Dunaliella maritima );(ii)the Ca 2+uptake de-pended on the intracellular Na +release;and (iii)the agents blocking the Ca 2+channels did not affect the transport of Ca 2+and Na +.

The Ca 2+ionophores A23187and ionomycin could enhance the Ca 2+in?ux by shuttling Ca 2+across biological membranes at stoichiometries of 2:1(A23187/Ca 2+)and 1:1(ionomycin/Ca 2+)(Liu and Hermann 1978,Hable et al.2001).For example,Hable et al.(2001)used A23187and ionomycin to increase drastically the cytosolic Ca 2+activity in an analysis of the effects of Ca 2+ionophores on the early development of fucoid algae.In another study,compared with the transient increases in the intracellular Ca 2+level that were induced by NaCl and sucrose,the Ca 2+ionophore A23187enhanced the Ca 2+in?ux further in Anabaena sp.PCC7120(Torrecilla et al.2001).However,in the present study,ionomycin did not have an effect as a Ca 2+ionophore to enhance the Ca 2+in?ux,possibly as a result of the impenetrable cell wall of Chlorella ;therefore,the Ca 2+iono-phore may be unsuitable to induce an extracellular Ca 2+in?ux into the cytoplasm in Chlorella sp.C2.

The comparison of the changes in the cytosolic Ca 2+level and intracellular neutral lipid content showed that the cytosolic Ca 2+level in each treatment nearly corresponded to the neutral lipid accumulation (Figs.2–6),which further demonstrates the regulation of Ca 2+signal transduction during neutral lipid syn-thesis in Chlorella sp.C2.However,the blocking effects on the cytosolic Ca 2+levels (VP GdCl 3)in the cells that were pre-treated with VP and GdCl 3at ODFs and LDFs,suggesting that other signal factors or a non-Ca 2+-mediated signal pathway for the regulation of neutral lipid synthesis may co-exist in Chlorella sp.C2under N starvation.For ex-ample,a study by de Montaigu et al.(2010)suggested that a nitric oxide (NO)-dependent guanylate cyclase gene CYG56participated in ammonium-mediated nitrate reductase repres-sion in Chlamydomonas through a pathway that involved the signal factors NO,cGMP and calcium,and that

similar

Fig.5Representative confocal laser scanning micrographs of Chlorella sp.C2cells labeled in vivo with BODIPY 505/515.CLSM images of cells with BODIPY 505/515?uorescence (green)and Chl auto?uorescence (red)in each treatment were recorded at 0,0.5,2and 8d after N starvation.N–,N starvation;RR,ruthenium red;VP,verapamil;Gd,GdCl 3;Ion,ionomycin;N +,N suf?cient.All of the ?gures are repre-sentative of three replicated studies with similar ?ndings.The size of the scale bar is shown directly on the image.

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mechanisms might occur in plants.Another study also revealed a copper-induced cross-talk among the signal factors calcium,H 2O 2and NO in Ulva compressa (Gonzalez et al.2012).

Perception of stress cues to excite Ca 2+signals and relay of the signals to switch on adaptive responses are the key steps leading to plant stress tolerance (Chinnusamy et al.2004).When Chlorella sp.C2was exposed to N starvation,the envir-onmental stimuli might be recognized by membrane sensors and then activate the Ca 2+channels in the PM and the mem-branes of intracellular calcium stores through a series of phos-phorylation reactions,all of which resulted in rapid rises in the Ca 2+level in the cytoplasm derive both from the extracellular space and from intracellular calcium stores to excite Ca 2+

signals (Figs.1–3).The Ca 2+signals might be translated to the downstream pathways via interactions with calcium-bind-ing proteins,and a series of biochemical reactions (e.g.neutral lipid synthesis)were induced and regulated to respond to N starvation (Figs.4–7).However,the N starvation stress sensors are not known and most of the signaling intermediates have not been identi?ed,so the studies on Ca 2+signal transduction pathway in microalgae under nitrogen starvation conditions are signi?cant and will be carried out step by step in the future.In summary,N starvation induced a transient increase in the cytosolic Ca 2+levels in Chlorella sp.C2,and the algal cells then maintained a stable,high cytosolic Ca 2+level during N starva-tion.Both the in?ux of Ca 2+from the extracellular space via

the

Fig.6FCM analysis of Chlorella sp.C2cells labeled in vivo with BODIPY 505/515.The ?uorescence of BODIPY 505/515in the cells (>104)was detected at 0d (a),0.5d (b),2d (c)and 8d (d)after N starvation.N–,N starvation;RR,ruthenium red;VP,verapamil;Gd,GdCl 3;Ion,ionomycin;N +,N suf?cient.All of the curves are representative of three replicated studies with similar

?ndings.

Fig.7Effect of Ca 2+channel blockers under N starvation on the total lipid contents in Chlorella sp.C2.The total lipid contents were detected,and the relative value of the lipid content in each sample compared with the control (N–)was calculated at 2d (a)and 8d (b)after N starvation.N–,N starvation;RR,ruthenium red;VP,verapamil;Gd,GdCl 3;Ion,ionomycin;N +,N suf?cient.The relative content of “1.0”represents the actual total lipid contents in cells under N starvation are 4.23%and 29.24%of dry cell weight at 2d (a)and 8d (b).The columns represent the means of three replicated studies in each sample,with the SD of the means (t -test,P <0.01).The signi?cance of the differences between the control (N–)and test values was tested using a one-way ANOVA.*P <0.05vs.control.

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Ca 2+channels that are localized to the PM and the release of Ca 2+from the intracellular calcium stores via the internal cal-cium store Ca 2+channels resulted in the increase in the cyto-solic Ca 2+levels that was required for the generation and transduction of the Ca 2+signal.By regulating lipid,in particular neutral lipid,synthesis,Ca 2+signal transduction pathways played signi?cant roles in the regulation of the stress response mechanism of Chlorella sp.C2under N starvation.In addition,different Ca 2+channels produced different regulatory effects on the stress response,and there might be a synergistic effect of the Ca 2+in?ux via all of the Ca 2+channels or another Ca 2+in?ux mechanism of Ca 2+signal transductions.Another non-Ca 2+-mediated signal pathway may also exist in Chlorella sp.C2in response to N starvation.

Materials and Methods

Growth conditions and N–treatment

The N-suf?cient (N +)medium that was used was full-strength BG11medium (Stanier et al.1971).The N-de?cient (N–)medium was BG11medium without NaNO 3.Chlorella sp.C2in the exponential phase was used to inoculate at an initial OD 700of 0.05(about 1.1?106cells ml –1)into a 1liter Erlenmeyer ?ask containing 500ml of BG11medium at 20 C with continuous illumination of 70m mol m –2s –1,continuously bubbled with ?ltered air.The cells were harvested by centrifu-gation at 6,000?g for 3min at 20 C at the mid-logarithmic growth phase (OD 700approximately 0.8,about 1.1?107cells ml –1)and were then washed and resuspended in N–medium to an OD 700of approximately 0.7(about 9.3?106cells ml –1).

Ca 2+?ux measurements

The net ?ux of Ca 2+was measured non-invasively using the SIET (the SIET system,BIO-001A,Younger USA Sci.&Tech.Corp.,Applicable Electronics Inc.and Science Wares Inc.).The record-ings of the Ca 2+?ux were performed as described by Sun et al.(2010)with some modi?cations.For the Ca 2+?ux recordings,the control or N–Chlorella sp.C2cells were settled on the center of a poly-L -lysine-pre-treated cover slip and then placed in 4ml of measuring solution (0.18mM K 2HPO 4á3H 2O,0.3mM MgSO 4á7H 2O,0.24mM CaCl 2and 0.19mM NaCO 3,pH 7.1).The cell populations (approximately 10cells)of Chlorella sp.C2on the cover slip were selected for the Ca 2+?ux measure-ment,which was continuously recorded for 5min.Three-dimen-sional ionic ?ux signals were plotted with MageFlux software that was developed by Yue Xu (https://www.360docs.net/doc/fc5787783.html,/mage?ux).

Pre-treatment with Ca 2+channel blockers and a Ca 2+ionophore

Before the N–treatment,three Ca 2+channel blockers and a Ca 2+ionophore were added to some of the Chlorella sp.C2cells and pre-incubated for 1h.These channel blockers included a stretch-activated Ca 2+channel blocker GdCl 3,a voltage-

dependent Ca 2+channel blocker VP,and a putative mitochon-drial and endoplasmic reticulum Ca 2+channel blocker RR,whose ?nal concentrations were 0.2mM,20m M and 20m M,respectively.The ?nal concentration of the Ca 2+ionophore ionomycin was 2m M.

Fluorescence imaging of cytosolic Ca 2+

For the ?uorescence imaging of the cytosolic Ca 2+,the Chlorella sp.C2cells were loaded with a Ca 2+-sensitive ?uorescent dye,Fluo-3AM,according to Chen et al.(2011).The ?uorescence images of the cytosolic Ca 2+from the Chlorella sp.C2cells that were loaded with Fluo-3AM were obtained using the Ratio Fluorescence Imaging System (EasyRatioPro),and the change in the ?uorescence of the cytosolic Ca 2+in a single cell was also recorded.The excitation and emission wavelengths were 488and 525nm,respectively.The ?uorescence of the cytosolic Ca 2+was continuously recorded for 5min.

Thin-layer chromatography lipid analysis

A 10ml culture at OD 700=1(about 1.3?107cells ml –1)was harvested at 6,000?g for 3min,and the cell pellet was washed with fresh medium and centrifuged again.The har-vested cell pellet was resuspended in 400m l of a metha-nol :chloroform mixture (1:1,v/v).The mixture was shaken for 2min,followed by phase separation using 120m l of 1M potassium chloride in 0.2M phosphoric acid.The mixture was then centrifuged at 12,000?g at room tempera-ture for 5min,and the chloroform phase was transferred to a glass tube and dried under nitrogen.The residue was re-suspended in a volume of 20m l of chloroform to obtain the lipid extracts.A TLC analysis of the lipid extracts from whole cells was performed according to Reiser and Somerville (1997)with some modi?cations.The triacylglycerols (TAGs)were separated by developing the plates in hexane :ethyl ether (7.5:2.5,v/v).The samples were visualized by exposure to iodine vapor for approximately 10min.Aliquots of 3m l for each sample were extracted at different time points and used for the TLC analysis.Glyceryl trioleate (3m l,10mg ml –1)was used as a reference substance for the TAGs,and the neutral lipid content of Chlorella sp.C2was then determined using ImageJ (v1.41,NIH)(Tsihlis et al.2010)and calculated as a value relative to the reference substance.

Confocal laser scanning microscopy analysis

A microscopic analysis of the cells was performed using a confocal scanner (Zeiss LSM 710NLO).The transmission micrographs that were used for the visualization of the non-?uorescent protoplast structures were generated using the manufacturer’s ?lter settings.A lipophilic ?uorescent dye,BODIPY 505/515(4,4-di?uoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-sindacene;Invitrogen Molecular Probes),was used to stain the intracellular oil-containing organelles,known as lipid bodies,with a ?nal labeling concentration of 1m M to-gether with 0.1%dimethylsulfoxide (DMSO;v/v)according to

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Cooper et al.(2010).The BODIPY?uorescence(green)was excited with an argon laser(488nm)and detected at 505–515nm.The auto?uorescence(red)of the algal chloro-plasts was detected simultaneously at650–700nm.

Flow cytometry analysis

The samples that were stained with Fluo-3AM or BODIPY 505/515were analyzed on a board using a FACSAria?ow cyt-ometer(Becton Dickinson)that was equipped with a laser emitting at488nm and an optical?lter FL1(530/30nm). The collected data were analyzed using FlowJo software(Tree Star).

Total lipid analysis

Cells at OD700=1(about1.3?107cells ml–1)were harvested by centrifugation at6,000?g for3min,and the cell pellet was washed with fresh medium and centrifuged again.Then the cell pellet was dried using a freeze dryer.Total lipid of micro-algae was extracted with methanol:chloroform(1:1,v/v)from 1g of lyophilized material and quanti?ed gravimetrically(Bligh and Dyer1959).

Statistical analyses

Each result that is shown is the mean of at least three biological replicates.A statistical analysis of the data was performed using the program SPSS-13,and the signi?cance was determined at the95%or99%con?dence limits.

Funding

This work was supported by the National Program on Key Basic Research Project[2011CB200902];the National Natural Science Foundation of China[31300030]. Acknowledgments

We thank Professor Xudong Xu for providing the Chlorella strain C2.

Disclosures

The authors have no con?icts of interest to declare.

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