Phospholipids

Abstract

Cytoskeleton serves as structural, membrane-bound and highly nonlinear dynamics element that basically functions in abiotic and biotic stresses. The study of phospholipid-regulated cytoskeletal organization to strengthen plants against stresses is emerging. Phospholipids in lipid bilayers, as the main compound of cellular membranes, have roles in modulation of membrane curvature and anchoring, cross-linking or regulating particular cytoskeletal proteins to modulate cytoskeletal dynamics. In this review, we highlight the role of phospholipids and their metabolic enzymes through regulating cytoskeletal organization and dynamics in response to abiotic stresses, such as salt, drought and low/high temperature stresses.

Introduction

Phospholipids are main components of cell membranes that are of lipid bilayer and chiefly sense environmental cues including biotic and abiotic stresses, which transduce external signaling into cells and make cells transform their morphology or physiological activities in order for them to adapt to new atmosphere. In recent years, increasing studies uncover phospholipids are vital mediators in plant processes, e.g., stomatal movement, root hair development, nutrient uptake, cytoskeletal dynamics, salt stress and heat shock.^1–12

In plant kingdom, the cytoskeletal system consists of microtubules (MTs) and microfilaments (MFs). MTs are composed of α- and β-tubulins, which form dimers and then polymerize into MTs. MFs are derived from G-actin polymerization. During plant growth, development and responses to adverse stresses, MTs and MFs dynamics are precisely regulated by both external and intracellular signals, where microtubule-associated proteins and microfilament-associated proteins participate. Generally, cytoskeleton often need reorient itself under stresses.^13–15 It has been well documented MTs and MFs take part in salt stress, heat shock, dehydration and drought.^{10,12,13,15–18} However, the exact mechanisms about how MTs and MFs sense cues and reorganize themselves in these processes remain to be elucidated.

Interestingly, phospholipids interacting with cytoskeleton and regulating its dynamics by affecting its polymerization or depolymerization directly or changing MTs and MFs-associated proteins activities, thereby resulting in cytoskeletal stability or destabilization, are studied and elaborated. Nevertheless, studies on which, whether and how phospholipids regulate cytoskeleton are still a bit few. In this review, we focus on the interaction between phospholipids and cytoskeleton, and highlight phospholipids-regulated cytoskeletal dynamics is of significant effector for plant growth and survival.

Phospholipids Shaping Cytoskeletal Organization under Salt Stress

It is widely accepted that phospholipids are versatile signal molecules, e.g., phosphatidic acid (PA), phosphatidylinositol (PI), inositol-1,4,5-bisphosphate (Ins(1,4,5)P₃) and lysophospholipids (LPs), involved in various stress processes including drought, salt, heat shock, etc.^9,10,19–22 Phospholipase D (PLD), which can hydrolyze phospholipids into phosphatidic acid and head group, is involved in plant salt tolerance. Arabidopsis PLDα1 and PLDδ are proved to function cooperatively in high salinity. Under high salinity, ablation of PLDα1 or PLDδ made plants more sensitive to salt stress, showing consistently lower PA levels than those of stressed wild-type plants as well as more severe inhibited root growth. The double mutant, pldα1pldδ, was shown to be the most sensitive to salt stress compared with regardless of wild-type or single mutants.²² By contrast, silencing of tomato (Lycopersicon esculentum) LePLDα1 did not render mutants any salt sensitivity. They showed the same root growth rate as wide-type plants. It may be accounted for incomplete ablation of LePLDα1.²² Hong et al. (2008) found that Arabidopsis PLDα3 positively regulate plant stress tolerance. Knockout of PLDα3 resulted in increased sensitivity to salinity and drought, while overexpression of PLDα3 aroused more resistance to these stresses.²³ Besides, PLDα1-derived PA was found to enhance plant salt tolerance by interacting with MPK6 (mitogen-activated protein kinase 6), a typical protein kinase proved to be involved in numerous processes.¹¹ More specifically, the pldα1 survived less under salt stress (≥ 150 mM NaCl) than wide-type plants. RNA interference (RNAi) and knockout of MPK6 gave rise to less tolerance for mutant plants. NaCl treatment activated PLDα1, which subsequently hydrolyzed membrane lipids into PA. PA bound to MPK6 and stimulated its activity. The elevated MPK6 phosphorylated C-terminal of SOS1 (Salt Overly Sensitive 1), a plasma membrane Na⁺/H⁺ antiporter, thereby enhancing salt tolerance of plants.¹¹

Cortical MTs play a vital role in determining growth axis and expansion of cells. Additionally, the array of cortical MTs is often changing when suffered from both development cues and stressed conditions. Keeping dynamic stability is essential for various cellular processes. In an early study, Dhonukshe et al. (2003) reported that short-term and high concentration NaCl treatment triggered rearrangement of cortical MTs in BY-2 cells.⁴ Recently, cortical MTs were verified to participate in the reaction to salt stress. Abolishment of SPIRAL1 (SPR1) caused right-handed helical growth with microtubule disruption, whereas, mutation of SOS1 and its regulatory kinase SOS2 in spr1 background by forward genetics reversed this phenotype, similar to that of wide-type plants. This suppression was specific to spr1 rather than other alleles mutants.¹⁶ In addition, SPR1 also adopt another way involving degradation of itself by 26s proteasome to regulate plants salt tolerance.¹² The salt stress induced a rapid depolymerization of MTs, and then remodeled a new MT network. Under salt stress, SPR1 was degraded rapidly by a 26s proteasome-RPN1a (Regulatory Particle Non-ATPase subunit 1a), thus leading to dynamic destabilization and fast disassembly of cortical MTs. After that, cortical MTs rearranged to new MT arrays, therefore improving plants salt tolerance,¹² which was consistent with a previous study where depolymerization and reorganization of cortical MTs are both of significant importance.¹³ Upon long-term salt stress, cortical MTs underwent depolymerization, and then reorganization. Using microtubule-stabilizing drug-paclitaxel evoked high death rate under salt stress. On the other hand, microtubule-destabilizing drugs, oryzalin or propyzamide, enabled seedlings to survive more under salt stress.¹³ Further investigation revealed that this process depended tightly on cytoplasmic calcium levels as removing calcium from the MS medium decreased survival rate under salt stress even in the presence of oryzalin.¹³ Although Dhonukshe et al. (2003) studied there was link between phospholipids and MTs, the underlying mechanisms that activation of PLD in BY-2 cells induces rapid MTs reorganization remain unclear.⁴ Afterwards, Zhang et al. (2012) first elucidated that PA interacted with microtubule cytoskeleton to regulate plants salt response in detail. More exactly, salt treatment activated PLDα1 followed by a rapid rise of PA. PA then bound to MAP65-1 (Microtubule-associated protein 65-1) through specific amino acids and enhanced its activity of polymerizing and bundling cortical MTs. Eventually, improvement of plants salt tolerance was achieved (Fig. 1).¹⁰

Figure 1. The phospholipid-cytoskeleton network in response to abiotic stresses. PI-PLC, phosphoinositide-specific PLC; DGK, diacylglycerol kinases; PC, phosphatidylcholine; PE, phosphatidylethanolamine; DAG, diacylglycerol; PtdIns(4,5)P₂, phosphatidylinositol 4, 5-bisphosphate; Ins(1,4,5)P₃, inositol 1, 4, 5-triphosphate; CP, heterodimeric capping protein; HSP, heat shock proteins; MTs, microtubules; MFs, microfilaments.

Conversely, unlike MTs behaviors in the responses to salt stress, plant MFs arranged salt responses by stabilizing MFs. 150 mM NaCl promoted MFs assembly and bundle formation, but high salt concentration-250 mM NaCl initiated MFs assembly and subsequently induced MFs disassembly. 250 mM NaCl is a lethal concentration, while 150 mM NaCl is a sublethal concentration that plants can withstand.¹⁴ The underlying different mechanisms maybe account for these processes, which still need to be uncovered further. Treatment with MF-disrupting drugs, latrunculin A (Lat A) and cytochalasin D (CD), resulted in more seedlings death under salt stress. In contrast, supplement with MF-stabilizing drug-phalloidin promoted seedlings more resistant to salt stress. Also, stabilization of MFs rescued survival rate of sos2 mutant.¹⁴ Taken together, stabilization of MFs enhances seedlings to survive in salt stress. Apart from SOS2, SOS3, another component of SOS signal pathway, guided MFs reorganization as well.²⁴ The hypersensitivity of sos3 to salt stress resulted from abnormal MFs arrays and dynamics. Loss of SOS3 function in sos3 changed MFs organization to bundle longitudinally to cell axis in the absence of NaCl, and NaCl treatment led to MFs depolymerize into fragments and arrange disorderly. However, exogenous supplement with calcium or Lat A partially rescued MF arrays to arrange well-organized in sos3, thereby reversing partially growth defects of sos3 under salt stress.²⁴ Interestingly, PLD and its derived product-PA have been proved to interact with MFs cytoskeleton and regulate MFs dynamics.²⁵ It was depicted PLDβ1-PA controlled MFs dynamics in tobacco pollen tube.²⁶ Most of pollen tube MFs depolymerized in the presence of n-butanol, which is a specific inhibitor of PA generation, and the depolymerized MFs could be recovered by addition of PA. NtPLDβ1 preferentially bound to MFs and G-actins, and its activity was enhanced by MFs, but was inhibited by G-actins, thus forming a positive feedback loop.²⁶ Indeed, PA could bind to the heterodimeric capping protein (AtCP) from Arabidopsis and inhibited its end-capping activity, leaving filament ends highly dynamic and allowing rapid assembly and elongation of MFs from free ends (Fig. 1).^25,27

Phospholipids Regulating Microtubular Arrays in Response to Drought

Accumulating evidence has pointed out that phospholipids are involved in the responses to drought, abscisic acid (ABA) and dehydration.^{17–20,28,29} Katagiri et al. (2001) first cloned a novel PLD cDNA that was named AtPLDδ by scanning the cDNA library from dehydrated Arabidopsis. The AtPLDδ regulated dehydration response in both transcriptional and posttranscriptional levels. Its mRNA accumulated rapidly and derived product-PA increased dramatically as well.²⁸ Moreover, PLDδ was reported as a candidate microtubule-associated protein.⁵ Additionally, the cellulose-interactive protein1 (CSI1) which is also a microtubule-stabilizing protein and regulates microtubule dynamics, was involved in regulation of dehydration. Dehydration changed colocalization between MTs and CSI1, promoting microtubule depolymerization and rearrangement.¹⁷ PLDα1-PA inhibited ABI1 phosphatase 2C activity by interacting with ABI1 and regulating its spatial localization in response to ABA.¹⁹ Besides, there are some other pathways involving PLD-PA having been reported to transduce ABA signaling. Specifically, PLDα1-derived PA bound to respiratory burst oxidase homolog D (RbohD) and stimulated its activity, followed by production of massive reactive oxygen species (ROS) to induce stomatal closure.²⁹ Indeed, disruption or stabilization of MTs would trigger maize root cells to biosynthesize ABA in maize root cells, which would regulate gene expression at the transcriptional level in response to external osmotic stress.³⁰

Formins (FH2 proteins) are known to be morphoregulatory proteins that modulate the assembly of unbranched actin filaments in eukaryotes.³¹ Plant formins are grouped into 2 subfamilies, type I and type II. Type I associated with the plasma membrane with an N-terminal transmembrane domain, whereas this feature is absent from type II formins.³¹ Plant formins are good candidates for regulators of cortical actin and microtubule cytoskeletons with the plasma membrane to mediate cell and tissue morphogenesis.^32–34 A type II formin from moss plants was reported to bind to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) through its N-terminal phosphatase and tensin (PTEN) domain. The interaction of PTEN domain and PtdIns(4,5)P₂ is required for cortical targeting of formin and polarized growth.³⁵

Microtubule arrays are required for normal guard cell function by changes in microtubule clustering or bundling, but the role of the microtubule cytoskeleton during stomatal movements is still controversial.³⁶ Jiang et al. (2013) described PA induced stomatal closure by integrating calcium signal and microtubule arrays in response to ABA in Arabidopsis. Under ABA treatment, cytoplasmic calcium elevated and activated AtPLDα1 to produce PA. Then PA induced stomatal closure by microtubule-dependent or independent pathway.¹⁸ Altogether, PA may regulate microtubule dynamics to transduce ABA, drought signaling by specific mechanisms, which still need to be elucidated.

Both the actin-related protein 2/3 (ARP2/3) complex and SCAB1 (Stomatal closure-related actin binding protein) are involved in the precise regulation of actin organization and stomatal closure in response to ABA.^37–39 H₂O₂-induced stomatal closure was delayed in arpc4 and arpc5 mutants, and the rate of actin reorganization decreased in arpc4 and arpc5 under H₂O₂ treatment, suggesting that ARP2/3-mediated actin nucleation is required for H₂O₂-induced actin cytoskeleton organization.³⁹

Cytoskeleton Integrating Phospholipids Signal into the Response to Low/High Temperature

Although there are some researches on MTs regulating plants heat shock response, the mechanisms involved in this process remain ambiguous. Depolymerization and reorganization of MTs are essential for plants to survive in heat shock stress. Upon short-term heat shock, MTs depolymerized transiently, and then repolymerized till full recovery at room temperature.¹⁵ In the meantime, depolymerization of MTs could also trigger calcium influx.^40,41 The rised calcium activated a 44 kDa heat shock-activated MAPK (HAMK) belonging to MAPK cascades upon heat stress.^42,43 Similarly, using MFs-disrupting drugs, Latrunculin B, also triggered extracellular calcium influx, followed by activation of HAMK, whereas that activation was blocked by MFs-stabilizer, jasplakinolide. Heat shock proteins (HSPs) are highly conserved molecular chaperone helping protein fold accurately in eukaryotic kingdom. Two HSPs, namely HSP70 and HSP90, can bind to MTs.^44,45 The binding between HSP90 and MT was demonstrated both in vitro and in vivo. Inhibition of HSP90 led depolymerized MTs to fail to repolymerize.⁴⁴ HSP70 was isolated from ATP-dependent microtubule-associated protein (ATP-MAP) complexes in tobacco cells. HSP70 binding to MTs was augmented in the presence of ATP and a 90-kDa Kinesin.⁴⁵ Heat shock activated PLD and phosphatidylinositolphosphate kinase (PIPK), and accompanied by PA and PtdIns(4,5)P₂ dramatic rise. PA and PtdIns(4,5)P₂ then targeted downstream partners to transduce signaling in response to heat stress.²⁰ Phosphoinositide-specific phospholipase C9 (PI-PLC9) was also reported to participate in heat shock response in Arabidopsis. Heat shock triggered accumulation of inositol-1,4,5-trisphosphate (InsP₃), followed by cytoplasmic calcium increase in wide-type, but was both abolished in atplc9 mutant.⁹ Interestingly, PLDδ, a candidate MAP, was found to associate with 5 HSP70s (i.e., HSP70-1, HSP70, HSP70-2, HSP70-3 and HSP70T-1) via in vivo co-immunoprecipitation.⁷ Therefore, PLDδ may regulate heat shock response by interacting with HSPs and MTs. Furthermore, MFs are also been revealed to modulate Arabidopsis thermotolerance. AtCPA and AtCPB were both upregulated from both transcription and protein levels under heat shock.⁴⁶ Surprisingly, only the atcpß mutant possessed high thermotolerance instead of atcpα mutant. In the atcpß mutant, there were more intact MFs compared with wide-type plants upon heat shock. Because of destabilization effects on MFs conferred by AtCPA and AtCPB, it is predicted readily that knockout of AtCPB results in mutants harboring more stable MFs, thus plants can withstand heat stress.⁴⁶

Although little is known about the cytoskeletal dynamics caused by low temperature upstream or downstream of the response to cold, it is believed that low temperature will cause depolymerization of MTs and MFs.⁴⁷ Both MTs and MFs in tobacco BY-2 interphase cells disassembled or disappeared after 20 min of 0 ^◦C treatment.⁴⁸ Transient depolymerization of MTs is sufficient for an efficient induction of acclimation in winter wheat with downregulation of α-tubulins, suggesting MTs act as efficient sensing of low temperature, culminating in the induction of acclimation machinery.⁴⁹ At the same time, membrane phospholipid metabolism also plays an important role in signaling cold responses. Knockout of PLDδ resulted in plants more sensitive to freezing, whereas overexpression of PLDδ enhanced freezing tolerance. Further study demonstrates that the PLDδ promotes freezing tolerance by increasing generation of PA species.⁵⁰ Interestingly, MAP65–1 is also reported to protect MTs against cold-induced depolymerization in vitro, which is regulated by PA derived from PLDα1 under salt stress.^10,51 This suggests that plant cells may have differential ability of incorporating internal phospholipids and different response machinery when exposed to different stresses.

Conclusions

Dynamic cytoskeleton changes are thought to be beneficial for plants to survive under stress conditions. Membrane is mainly composed of phospholipids which are hydrolyzed by multiplex phospholipases. Composition of membrane often changes by phospholipases, thereby affecting fluidization of membrane or producing signaling phospholipids in response to external or intracellular stimuli. Cortical MTs and MFs are localized underneath the plasma membrane. Some linkers between plasma membrane and cytoskeleton, such as CSI1, formins, and PLD are reported. However, the molecular mechanism for interaction of phospholipids-cytoskeleton is still limited.⁵² For example, PLDα1-derived PA binds to and regulates MAP65-1 to stabilize MTs in response to salt stress.¹⁰ However, there is no direct evidence to determine how PA regulates microtubule dynamic instability. Does PA increase polymerization rates at (+) ends, or slow depolymerization from (–) ends? And do the products of PA, diacylglycerol (DAG) and diacylglycerol pyrophosphate (DGPP), also regulate the cytoskeleton? Moreover, PtdIns(4,5)P₂ also bind to AtCP and inhibit its capping activity except PA.^25,27 What are the molecular effect of PtdIns(4,5)P₂ on microfilament organization to enhance tolerance under abiotic stresses? Therefore, in future work, it is necessary to further clarify more phospholipids, including PA, DAG, PtdIns(4,5)P₂ and InsP₃, and their binding or interacting cytoskeletal proteins upon stresses.