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(The FASEB Journal. 2009;23:623-630.)
© 2009 FASEB

Lipidomic analysis reveals activation of phospholipid signaling in mechanotransduction of Taxus cuspidata cells in response to shear stress

Pei-pei Han and Ying-jin Yuan1

Key Laboratory of Systems Bioengineering, Ministry of Education and Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China

1Correspondence: Key Laboratory of Systems Bioengineering, Ministry of Education and Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China. E-mail: yjyuan{at}tju.edu.cn or yjyuan{at}public.tpt.tj.cn


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipid signaling involved in mechanotransduction processes in response to shear stress in plants remained elusive. To understand the responses of phospholipids in shear stress-induced mechanotransduction, a lipidomic approach was employed to profile phospholipid species of Taxus cuspidata cells under laminar shear stress. A total of 99 phospholipid species were profiled quantitatively, using the LC/ESI/MSn procedure. Potential biomarkers were found by the principal component analysis (PCA) as well as partial least squares (PLS) combined with variable influence in the projection (VIP). Phosphatidic acid (PA) and lysophosphatidylcholine (LysoPC) were two important lipid groups that were responsible for the discrimination between shear stress induced and control cells. Further research revealed that shear stress enhanced the activation of phospholipase D (PLD) and phospholipase C (PLC) compared with control cells and consequently increased PA content in shear stress induced T. cuspidata cells. These results demonstrate that phospholipids and related phospholipases play important roles in mechanotransduction of T. cuspidata cells in response to shear stress.—Han, P.-P., Yuan, Y.-J. Lipidomic analysis reveals activation of phospholipid signaling in mechanotransduction of Taxus cuspidata cells in response to shear stress.


Key Words: phospholipase • phosphatidic acid • mechanical stress • plant cell culture • metabolomics • LC • MS/MS


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE MECHANOTRANSDUCTION PROCESSES in response to shear stress, by which cells convert mechanical stimuli into biochemical signals, have gained increasing attention. Shear stress is a fundamental determinant of vascular homeostasis, regulating vascular remodeling, cardiac development, and atherogenesis (1) , and it has been widely investigated in animal cells. Plants can also sense and respond to mechanical stimuli, like animals. Mechanotransduction is a complex process, involving the participation of a multitude of sensors, signaling molecules, and genes. A number of mechanosensitive biological molecules in animals have been identified, including mechanically gated channels, receptors, G proteins, enzymes, and cytoskeleton (1 2 3 4) . However, the study of molecular mechanisms of mechanotransduction in plants is far behind similar studies in animals, and the mechanotransduction processes in plants remain elusive.

At the cellular level of plants, signaling molecules, including membrane proteins, intracellular calcium, reactive oxygen species (ROS), mitogen-activated protein (MAP) kinase, and hormones respond to mechanical stimulation (5 6 7 8 9) . Our previous studies showed that RGD (Arg-Gly-Asp)-binding proteins located in cell membrane played a key role for Taxus cuspidata cells in reception to shear stress (10) . The roles of oxidative burst and NO generation were also investigated in our previous studies (11 , 12) . Lipids have long been recognized as signaling molecules that have the capacity to trigger profound physiological responses. However, the study of lipid signaling is a relatively young, up-and-coming field of research. Phospholipids, in addition to their structural role, play a much broader role; they can be signal molecules or signal precursors, or they may act as essential cofactors for membrane enzymes (13) . Typically, phospholipid signaling systems are grouped according to the phospholipases that initiate the formation of the messenger molecules. The products of phospholipase A, C, or D (PLA, PLC, or PLD) activity have been implicated in signaling cascades (14 15 16) . It was found that mechanical stimuli activate mammalian target of rapamycin (mTOR) signaling through a PLD-dependent increase in phosphatidic acid (PA) in skeletal muscle (17) . Yet, the function of membrane phospholipids in mechanical signal transduction in plants has not gained enough attention.

Toward this end, global molecular profiling methods hold great promise, as they provide a relatively unbiased portrait of the biochemical composition of cells and can reveal unanticipated alterations in their metabolic and signaling networks. Proteomics has been introduced to study mechanotransduction in response to shear stress (18) . Lipidomics, a subdiscipline of metabolomics, is emerging as a strategy that allows the systems-level analysis of lipids and their interacting partners. One of its advantages over traditional methods, such as thin layer chromatography (TLC), is that it allows quantitative analysis of a broad-range of lipid species in a single platform (19) . We have developed a LC/ESI/MSn based lipidomic approach and have identified more than 90 phospholipid molecular species in T. cuspidata cells (20 , 21) .

In this study, a lipidomic approach combined with multivariate analysis was employed to investigate the role of phospholipids in mechanotransduction of T. cuspidata cells in response to laminar shear stress (LSS). Large-scale cultivation of T. cuspidata cells has been developed as a promising alternative to produce the secondary metabolite paclitaxel, an anticancer drug (22 23 24) . However, the hydrodynamic shear stress has great influence on cell growth and production of secondary metabolites. A fuller understanding of the mechanism of shear sensitivity may assist optimization of large-scale cultivation conditions and regulation of physiological and pathological response to mechanical forces of cells by using molecular biological method. Here our study shows that lipidomics is a potential powerful strategy to investigate mechanotransduction processes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Sodium salt, 1, 2-dimyristoyl-sn-glycero-3-phosphate (14:0/14:0 PA); ammonium salt solution from bovine liver, L-{alpha}-phosphatidylinositol (PI); sodium salt, 1, 2-dipalmitoyl-sn-glycero-3-phospho-L-serine (16:0/16:0 PS); sodium salt, 1, 2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (14:0/14:0 PG); 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (14:0/14:0 PE); 1,2-dimyristoyl-sn-glycero-3-phosphocholine (14:0/14:0 PC); and 1-heptadecanoyl-sn-glycero-3-phosphocholine (17:0) were purchased from Sigma (St. Louis, MO, USA). Sodium salt, 1,2-dipalmitoyl-sn-glycero-3-phosphobutanol (16:0/16:0 PtdBut) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). HPLC-grade methanol was purchased from Merck (Darmstadt, Germany). HPLC-grade chloroform was purchased from J. T. Baker (Phillipsburg, NJ, USA). Ammonium hydroxide (28–30%) was purchased from J and K Chemical (Beijing, China). Neomycin and 1-butanol were obtained from Sigma.

Cell culture
The cell line from young stems of T. cuspidata was subcultured on a modified B5 solid medium (containing sucrose 25.0 g/L, {alpha}-naphthylacetic acid 2.0 mg/L, and 6-benzyladenine 0.15 mg/L) at 25°C in dark. Cell suspensions were subcultured at 25°C in dark with shaking at 110 rpm every 10–12 days, for a total of 5 generations, in fresh modified B5 medium. The pH of the medium was adjusted to 5.8.

Shear apparatus and experimental procedures
The Couette-type shear reactor was designed and built by Synthecon (Houston, TX, USA). The cylinders were arranged horizontally rather than vertically, as in traditional Couette viscometers (Fig. 1 ). The shear reactor was composed of two coaxial cylinders, the diameters of which were 45 and 53 mm, respectively. The static outer cylinder was transparent, which was convenient for monitoring the cell state. A gas-permeable membrane covered the outer surface of the inner cylinder and served as the oxygen exchanger to the culture medium. The plant cell suspension completely filled the 160 ml working volume of the bioreactor that lay in the annular chamber between the concentric outer and inner cylinders. Fluid dynamic shear stress, imposed by the rotation rates of the inner core, was the only physical factor affecting cell behavior.


Figure 1
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  		Figure 1. Schematic representation of a Couette-type shear reactor for studying shear stress effects on suspension cell cultures of Taxus cuspidata. The rotation speed of the inner cylinder was computer controlled.

Laminar shear rate {gamma} was estimated approximately according to the simplified equation (25) :

Formula
where {Omega} is the angular velocity, and K is the ratio of the inner to outer cylinder radius. The outer cylinder was kept static, and the inner cylinder was rotated at 100 rpm during experiments. The shear rate was 75 s–1 according to the above equation.

The shear reactor was sterilized with 70% alcohol before inoculating, and the temperature was maintained at 25°C. The suspension sample of T. cuspidata was loaded directly into the shear reactor after 8 days of subculture, when it was in the exponential growth phase.

Lipid extraction
Total lipid extraction of T. cuspidata cells was reported previously (20 , 21) . Briefly, fresh cells (~0.6 g) were immediately placed in 3 ml isopropanol with 0.01% butylated hydroxytoluene (BHT) at 75°C. Appropriate amounts of mixed internal standards were added during lipid extraction. The tubes were incubated at 75°C for 15 min, and 1.5 ml chloroform and 0.6 ml ultrapure water were added. The tubes were shaken for 1 h, followed by removal of the extract. The cells were reextracted with chloroform/methanol (2:1) containing 0.01% BHT 5 times, with 30 min of agitation each time. The combined extracts were washed once with 1 ml 1 M KCl and once with 2 ml ultrapure water. The solvent was then evaporated under nitrogen and the resulting lipid samples were stored at –20°C. The remaining cells were heated overnight at 105°C and weighed. Prior to analysis, the extracted lipid samples were redissolved in 2 ml chloroform/methanol (1:1, v/v).

Analysis of phospholipids by LC/ESI/MSn
Analysis of phospholipids was performed on a Waters LC-MS system, consisting of a 600E HPLC with an autosampler coupled with Quattro Micro API triple-quadruple mass spectrometer (Micromass, Manchester, UK), as described below.

A Kromasil 5µ Sil HPLC column (150x4.6 mm internal diameter, 5 µm particle size; Azko Nobel, Beijing, China) was used for separation. Gradient elution mobile phase consisted of A (chloroform:methanol:30% ammonium hydroxide=89.5:10:0.5, v/v/v) and B (chloroform:methanol:30% ammonium hydroxide:water=55:39:0.5:5.5, v/v/v/v). The following linear gradient was used: 5–30% B for 7 min, 30% B for 3 min, 30–50% B for 30 min, 50–95% B for 6 min, 95% B for 7 min, 95–100% B for 2 min, 100% B for 5 min, 100–5% B for 5 min, and 5% B for 5 min. The mobile phase was set at 1 ml/min. The injection volume was 10 µl.

The eluent from the HPLC column was split with 0.2 ml/min flow and directed into the ion source of the mass spectrometer. The Quattro Micro triple-quadruple mass spectrometer was operated in negative ESI ion mode, with a capillary voltage of 3.0 kV. The Quattro mass spectrometer was operated in full scan mode, scanning from m/z 500–1000 with a scan time of 500 ms and an interscan delay time of 100 ms, resulting in ~100 scans/min. The source temperature was set at 100°C and the voltage of cone, extractor and RF lens were 30, 3, and 0.1 V, respectively. Nitrogen was used as the desolvation gas and cone gas. Cone and desolvation gas flows were set at 400 and 50 L/h respectively, with desolvation temperatures at 350°C.

Data normalization and multivariate analysis
To identify the possible changes of phospholipids, quasi-quantification of various phospholipids by normalizing the peak area of individual molecular species with that of an internal standard was carried out. Principal component analysis (PCA) and partial least squares (PLS) combined with variable influence in the projection (VIP) scores were performed using the Matlab 7.0 (Mathworks, Inc., Natick, MA, USA) software package with mean-center scaling preprocessing. In the PLS model, the VIP index is introduced to quantitatively estimate the contribution of each metabolite variable to the pattern recognition (26 , 27) ; 8421 codes were used as label codes.

PLD assay and PLC assay
The in vivo activity of PLD was assayed by the formation of phosphatidylbutanol (PtdBut), a specific product of PLD in the presence of butanol. PtdBut was determined as reported previously (20) . T. cuspidata cells were incubated with 0.5% (v/v) 1-butanol for 4 h before the extraction of lipids. The lipids were analyzed by LC/ESI/MSn as described above. PtdBut molecular species were identified according to their retention times, molecular ions, and negative ion fragments.

The activity of PLC was assayed by adding neomycin, the inhibitor of PLC. Cells were incubated with 200 µM neomycin (dissolved in sterilized water) for 30 min prior to shear. Lipids were extracted and analyzed by LC/ESI/MSn as described above.

Statistics
The experimental data were the means of at least 3 independent experiments. The significance of differences between experimental points was determined by 2 independent sample t tests. A value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phospholipid profiles of T. cuspidata cells under LSS by LC/ESI/MSn
The LC-MS-based phospholipid profiling method has been published previously (20 , 21) . Here, it was used to analyze the response of cultured T. cuspidata cells to LSS. Fig. 2 shows the negative-ion LC/ESI/MS base peak chromatogram of phospholipids of LSS-induced T. cuspidata cells. Phosphatidylglycerol (PG) was firstly eluted, followed by phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI)/phosphatidylserine (PS) (PS partially coeluted with PI), PA and lysophosphatidylcholine (LysoPC). Most anionic and weak anionic phospholipids, such as PA, PI, PS, PG, and PE, were readily detected with [M–H]– as their molecular ions in ESI negative mode. The PC and LysoPC classes, having a choline group at the polar head, were detected as chloride adducts [M+Cl]–. Using the LC/ESI/MSn procedure, 99 phospholipid species were completely profiled quantitatively.


Figure 2
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  		Figure 2. Negative-ion LC/ESI/MS base peak chromatogram of phospholipids from T. cuspidata cells 40 h after LSS treatment (75 s–1). Peak identification is as labeled. IS indicates internal standard. ISPI and ISPS coelute with PI and PS. ISLysoPC partially coelutes with LysoPC.

Compared with T. cuspidata cells cultured in shake flask, the amounts of phospholipids in LSS-induced cells changed with time, as shown in Fig. 3 . The amounts of total PA and LysoPC increased, whereas the amounts of total PG, PE, and PC declined significantly (P<0.05). The amount of PI first decreased and then returned to its original level. In contrast, the amount of PS was not significantly different.


Figure 3
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  		Figure 3. Phospholipid contents in LSS-induced Taxus cuspidata cells. Data represent means ± SD of 5 independent experiments. *P ≤ 0.05 vs. control; t test.

Determination of metabolite changes by PCA and PLS
To investigate the metabolic differences of phospholipid species between LSS-induced and control cells, a PCA model was established to analyze the data. PCA was an unsupervised clustering method and was employed to gain insight into the nature of the multivariate data. PCA identifies and ranks major sources of variance within data sets and allows clustering of biological samples into both expected and unexpected groups based on similarities and differences in the measured parameters. Projection of the resulting sample scores for the first and second principal components (Fig. 4A ), which together accounted for 92.6% of the total sample variance, clearly separated the 5 experimental groups. The separation was mainly due to PC1, which accounted for 87.9% of the variability in the data. The PC1 spaces at the 48 h time point were completely different from those of control cells. Compared with other time points, the 48 h time point was most distinguished from the control group by PC1. Examination of the PCA loading plot (Fig. 4B ) revealed that the main variables responsible for separation were PA and LysoPC, which positively correlated in the PC1 space. Most of the other phospholipid species were near the origin, suggesting that their contribution to metabolic differences was less than that of PA and LysoPC.


Figure 4
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  		Figure 4. Multivariate analysis by PCA and PLS. A, B) Score plot (A) and loading plot (B) generated by PCA. C) Score plot generated by PLS. D) VIP plot from PLS analysis using 8421 codes. X-axis represents each phospholipid molecular species.

To further validate the difference between the control and LSS-induced cultured cells, PLS analysis, a supervised clustering method, was performed. The flexibility of the PLS approach, its graphical orientation, and its inherent ability to handle incomplete and noisy data with many observations make PLS a simple but powerful approach for the analysis of data of complicated problems. Examination of the score plot generated from PLS (Fig. 4C ) showed that LSS-induced cells were distinguished from control cells. To identify the most influential metabolites that characterize different response patterns, the VIP index was introduced. Figure 4D shows that PA and LysoPC were mainly responsible for discriminating between LSS-induced and control cells, and demonstrates that results obtained from PCA and PLS are in good agreement.

PA and LysoPC were concluded to be the most influential phospholipids that were responsible for the separation between LSS-induced and control cells by PCA and PLS. Their changes under LSS are shown in Fig. 5 . Compared with cells cultured in shake flask, LSS led to a significant increase in the PA and LysoPC species.


Figure 5
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  		Figure 5. Changes of biomarker phospholipids in LSS-induced T. cuspidata cells. A) PA. B) LysoPC. Data represent means ± SD of 5 independent experiments.

PtdBut formation in LSS-induced cells in the presence of 1-butanol
PLD hydrolyzes structural phospholipids, such as PC, to PA. PLD has the ability to transfer the phosphatidyl group from its substrate to a primary alcohol, such as 1-butanol, producing a phosphatidylalcohol such as PtdBut instead of PA. As the phosphatidylalcohol is not readily further metabolized, it can serve as a better marker for the assay of the in vivo activity of PLD than the formation of PA. The PtdBut species were easily detected by LC/ESI/MS2. It was shown (Fig. 6A ) that the amounts of PtdBut species in LSS-induced cells were higher than in control cells, thus demonstrating that PLD was activated by shear stress.


Figure 6
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  		Figure 6. Defining the enzymatic source of PA. A) Relative abundance of individual molecular species of PtdBut in LSS-induced T. cuspidata cells to control cells. B) Comparison of PA molecular species content in LSS-induced T. cuspidata cells 16 h with and without neomycin. Data represent means ± SD of 3 independent experiments.

Neomycin partly inhibits accumulation of LSS-induced PA
PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into 2 different second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG is rapidly phosphorylated by diacylglycerol kinase (DGK), generating PA (16) . To determine whether PLC was activated to generate PA during shear stress, we analyzed the effect of neomycin, a PLC/DGK pathway inhibitor that blocks synthesis of PA through inhibiting PLC activity, on the amounts of PA molecular species. Treatment with neomycin significantly inhibited accumulation of LSS-induced PA (Fig. 6B ). However, the amounts of PA species were still higher than in control cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mechanotransduction is a very complex process. Investigations on mechanosensitive biological molecules, such as mechanically gated channels, receptors, G proteins, integrins, enzymes, and cytoskeleton were broadly carried out (1 2 3 4 , 28 , 29) . Lipid signaling involved in the mechanotransduction process remains elusive. Previous studies on lipid-related signaling pathways have identified particular lipids that act to regulate the functions of a number of proteins, either by controlling enzyme activity directly or by localizing proteins to particular intracellular compartments, where they perform a specialized role (30) . The actual changes in lipid concentration in a stimulated cell are less characterized. The primary reason for this lack has been a deficiency in methodology. Radiocarbon labeling of endothelial cells has been used to study the changes of phospholipid after stimulation of fluid shear stress (31) . However, this type of methodology has many disadvantages, including lack of isotopic equilibrium, distinct pools with different turnover rates, and inadequate separation of radiolabeled metabolites (30) . Our work provided one case study introducing lipidomic strategy based on ESI-MS/MS into lipid signaling investigation.

The study of lipidomics, an important subdiscipline of metabolomics, has the potential to provide a comprehensive understanding of the influence of all phospholipids and related enzymes in biological systems (19 , 32 , 33) . This study shows that ESI-MS/MS allows rapid analysis of the composition of plant phospholipids. Here, more than 90 phospholipid molecules were profiled to investigate global differential changes in phospholipid composition following shear stress treatment. Application of multivariate statistical analysis such as PCA and PLS helps the identification of correlations in lipid metabolites that are associated with a physiological phenotype, in particular for the development of lipid-based biomarkers (19) . In our study, using PCA and PLS, differential changes in the abundances of phospholipids species were revealed in LSS-induced cells. The score plot showed that LSS-induced cells were distinctly clustered away from control cells, indicating that LSS had a significant effect on phospholipids.

Examination of loading plot and VIP plot revealed that PA was one important lipid group that discriminated between the LSS-induced and control cells. Evidence is accumulating that PA is a second messenger. Its level increases within minutes of a wide variety of stress treatments, including ethylene, wounding, pathogen elicitors, osmotic and oxidative stress, and abscisic acid (34) . Our previous studies revealed that PA molecular species mediate many important biological processes, such as mediating the apoptosis process of Taxus chinensis var. mairei cells and cell death response to Ce4+ in T. cuspidata cells (20 , 21) . Although it is not yet clear how PA functions in shear signaling pathways, interactions between PA and other signals are potential mechanisms. Our previous results of neomycin inhibiting generation of LSS-induced H2O2 suggested that PLC was involved in the signal pathway for oxidative bursts (11) . The present work revealed that PLC was also involved in the PA formation induced by LSS. We might presume there were some connections between PA and ROS signaling in the shear signaling network. Increases in PA in both tomato and Arabidopsis spp. have been linked to the initiation of ROS generation, a central event in plant resistance (35 36 37) . PA also represents a downstream component of NO signaling cascade during stomatal closure and in xylanase-elicited tomato cells (38 , 39) . In addition, PA could exert its effects through downstream signaling of MAPK cascade (40 , 41) . MAPK activation has been demonstrated to play key roles in stress responses of T. cuspidata cells (10) . Whether PA is correlated with these signal molecules in LSS-induced T. cuspidata cells requires further investigation.

Defining the enzymatic source of PA is important to understanding the cellular role of PA and cellular regulation of signaling pathways. It is generated via two distinct phospholipase pathways, either directly by PLD or by the sequential action of PLC and DGK.

The formation of relatively large amounts of PtdBut in LSS-induced cells suggested the activation of PLD occurred under LSS (Fig. 6A ). The identities of PtdBut molecular species provide information about the lipid substrates from which PtdBut species are derived. Therefore, we could get the information of preferred substrate for PLD, which showed one major advantage of the MS-based method over conventional methods, such as TLC (16) . The PtdBut profile closely matched the PC profile except for some minor long-chain polyunsaturated fatty acids, which were not detected in the corresponding PtdBut spectrum (Supplemental Table 1), indicating that PC is likely the preferred substrate for PLD in T. cuspidata cells. In addition, PE might in part serve as a substrate for PLD, since the PE profile also had some similarities to the PtdBut profile. PLD hydrolyzes the phosphodiester bond of the predominant membrane phospholipid, PC, producing PA and free choline. This activity has been shown to participate in signal transduction pathways (42) . Moreover, it was proved that mTOR signaling was activated through a PLD-dependent increase in PA by mechanical stimuli in skeletal muscle (17) .

Treatment with neomycin, an inhibitor of PLC, significantly inhibited accumulation of LSS-induced PA (Fig. 6B ), which demonstrated that PLC was activated by LSS. PA generated via the PLC/DGK-enzymatic pathway occurs in response to chilling, microbial elicitation, salt, and hyperosmotic conditions (43 , 44) . In addition, activation of the PLC/DGK pathway can produce many signal molecules besides PA, such as PIP2, IP3, and DAG. These all play important signaling roles in cells in response to environmental stress (45) . Furthermore, PLC itself is involved in many signaling processes. PLC was shown to be activated by collagen and antibodies to β2 integrins and was an important downstream component of integrin-mediated signal transduction (46) . Our previous studies indicated that an RGD recognition system might exist in T. cuspidata cells and play an important role in signal transduction of shear stress (10) . Thus, PLC might also play an important role in RGD-dependent mechanotransduction in LSS-induced T. cuspidata cells.

Analysis of the loading plot and VIP plot revealed that LysoPC was the other important lipid group that discriminated between the LSS-induced and control cells. LysoPC was reported to be an important signal in response to various stresses, such as in the arbuscular mycorrhizal symbiosis (47) . The levels of LysoPC species were increased by shear stress, which was most likely the result of activation of PLA. PC was hydrolyzed by PLA to release LysoPC and free fatty acids. In plants, PLA2 and its lysolipid product, LysoPC, have been reported to be involved in numerous cellular processes (15) , including cytoplasmic acidification, which is accompanied by extracellular medium alkalinization, with the latter leading to alterations in gene expression (40) . In addition, free fatty acids, concomitantly generated with LysoPC, are also important signal messengers. For example, LSS-induced cells exhibited increased LysoPC18:3 levels (Fig. 5 ), which was concomitant with an increase of linolenic acid (C18:3). In plants, the derivation of a phospholipid-derived acyl chain, linolenic acid, leads to the formation of oxylipins such as JA (48) .

In the LSS-induced cells, the amounts of structural phospholipids such as PG, PE, and PC were significantly decreased (Fig. 3 ). Hydrolysis of membrane phospholipids and increases in PA and LysoPC altered the organization and dynamic structure of membrane lipids and might eventually alter membrane physical properties, such as membrane permeability (49) . This might be one important reason for the increased membrane permeability caused by shear stress (Supplemental Fig. 1). Alterations in the bilayer physical properties can modulate shear stress activation in vascular endothelial cells (50) , and a similar signaling mechanism in response to shear stress might also exist in T. cuspidata cells.

In conclusion, using a lipidomic approach combined with multivariate analysis, we studied phospholipid profiles and revealed discriminatory phospholipid profiles between LSS-induced and control T. cuspidata cells. Our study showed that the activation of phospholipid signaling plays an important role in shear stress-induced mechanotransduction in T. cuspidata cells. Our work also demonstrated that lipidomics is a potential powerful tool to investigate mechanotransduction processes.


   ACKNOWLEDGMENTS
 
This work was funded by a National Science Fund of China for Distinguished Young Scholars grant (project 20425620), a Key Program grant (project 20736006), and the National Basic Research Program of China ("973" Program: 2007CB714301).

Received for publication August 8, 2008. Accepted for publication September 11, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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