sPLA₂ and PLA₁: Secretory Phospholipase A₂ and Phospholipase A₁ in Plants

Abstract

Plant phospholipase As (PLAs) are classified into two major types, PLA₁ and PLA₂, according to the hydrolysis sites of their membrane lipids. The lipid products released by PLAs have been suggested to act as bioactive molecules that mediate cellular signaling pathways functioning in plant growth and development, as well as responses to abiotic and biotic stimuli. The past few years have witnessed a wealth of new information regarding the function of these phospholipases in various biological processes. In this chapter, we discuss recent insights into lipid-based signaling mediated by PLAs and their lipid products, with particular emphasis on their emerging role as lipid mediators.

1 Introduction

Phospholipid-derived products generated by phospholipase A (PLA), such as free fatty acids (FFAs) and lysophospholipids, play critical roles in plants, as these products are the precursors of second messengers in signal transduction pathways. In addition, these signaling molecules function in important physiological processes such as cell elongation, gravitropism, anther dehiscence, biosynthesis of jasmonic acid (JA), and defense signaling (Chen et al. 2011; Ryu 2004; Wang et al. 2012). To understand the highly regulated production of these lipid mediators in response to diverse extracellular stimuli, it is important to study the functions and regulatory mechanisms of the diverse PLA enzymes. Recent developments in genetic and biochemical analysis have facilitated our understanding of the cellular functions of plant PLAs. The PLA superfamily has three subtypes, i.e., phospholipase A₁ (PLA₁), phospholipase A₂ (PLA₂), and patatin-like PLA (pPLA). PLA₁ and PLA₂ catalyze the hydrolysis of membrane glycerophospholipids at their sn-1 or sn-2 positions, respectively, while pPLA shows activity at both positions. In this chapter, we will describe the current classifications of PLA₁s and PLA₂s that have been identified in plants, especially in Arabidopsis, and recent advances in our understanding of how these PLAs are involved in various cellular signaling pathways.

2 Phospholipase A₂

Based on the functional, structural, and catalytic properties of lipolytic enzymes, the PLA₂ superfamily can generally be divided into five principal families in animals, categorized as secretory PLA₂s (sPLA₂), cytosolic PLA₂s (cPLA₂), Ca²⁺-independent PLA₂s (iPLA₂s), platelet-activating factor acetyl hydrolases (PAF-AHs), and lysosomal PLA₂s (Schaloske and Dennis 2006). Only low molecular weight secretory PLA₂s have been reported in plants, although patatin-like PLAs have been identified, which are similar to the iPLA₂s or cPLA₂ but exhibit both PLA₁- and PLA₂-like activity (Lee et al. 2005; Ryu 2004).

2.1 sPLA₂ Grouping, Expression, and Localization

Four and three secretory PLA₂ paralogs were identified in the Arabidopsis and rice genomes, respectively. Based on their primary structures, plant sPLA₂s are equivalent to animal group XI sPLA₂s, which are further divided into groups XIA and XIB. The former group includes AtsPLA₂-α, OsPLA₂-α, and OsPLA₂-β, while the latter group includes AtsPLA₂-β, AtsPLA₂-γ, AtsPLA₂-δ, and OsPLA₂-γ (Lee et al. 2005; Schaloske and Dennis 2006; Singh et al. 2012).

AtsPLA₂s exhibit different spatial and temporal expression patterns. AtsPLA ₂ -α and AtsPLA2-β are expressed in all sporophytic tissues. Unlike AtsPLA ₂ -α, AtsPLA ₂ -β is strongly expressed in actively growing young tissues and pollen. AtsPLA ₂ -γ and AtsPLA ₂ -δ are exclusively expressed in pollen. While AtsPLA ₂ -β is expressed during all stages of pollen development, AtsPLA ₂ -γ is expressed at low levels during the early stage of pollen formation and is strongly expressed at the mature stage, and AtsPLA ₂ -δ is expressed at the mature pollen stage (Bahn et al. 2003; Kim et al. 2011b; Lee et al. 2003).

Studies of the subcellular localizations of plant sPLA₂ have been performed using fused fluorescence proteins. These studies provide important insights into the functions of these proteins in plants. Early experiments involving transient expression analysis of green fluorescence protein (GFP) in onion epidermal cells showed that AtsPLA₂-β and AtsPLA₂-γ are secreted into the cell wall/extracellular space (Bahn et al. 2003; Lee et al. 2003), but this localization pattern is regarded as a result of the masking of the ER retention signal (KTEL) by that of the C terminal GFP fusion protein of AtsPLA₂-β. More recently, Seo et al. (2008) reexamined the subcellular localization of AtsPLA₂-β with N terminal GFP fusion protein of AtsPLA₂-β and observed that it indeed localizes to the ER in Vicia faba guard cells, and the result was supported in AtsPLA₂-β:YFP transgenic plants (Lee et al. 2010). Transient expression of AtsPLA ₂ -γ:YFP and AtsPLA ₂ -δ:YFP in tobacco leaf epidermal cells demonstrated that AtsPLA₂-γ localizes to the ER and Golgi, while AtsPLA₂-δ localizes to the ER and is found in the pollen of transgenic plants (Kim et al. 2011b). Subcellular localization of AtsPLA₂-α revealed a rather complicated localization pattern unlike that of its paralogs. An analysis of AtsPLA ₂ -α:DsRed2 transgenic Arabidopsis plants suggests that AtsPLA₂-α localizes to the Golgi in root tissues (Lee et al. 2010). In another study, AtsPLA₂-α exhibited different localizations in a time-dependent manner when this gene was introduced into Arabidopsis seedlings and tobacco leaves via Agrobacterium-mediated transient transformation (Froidure et al. 2010). The AtsPLA₂-α:YFP signal was detected in cytoplasmic vesicles around the nucleus 36 h after inoculation and was detected at the extracellular spaces outside of the cells at 48 h after inoculation. AtsPLA₂-α was partially localized to the cell nucleus when it was co-expressed with AtMYB30.

A more recent study shows that subcellular localization of AtsPLA₂-α is dependent on the developmental stage of the leaf tissue. Fluorescence signals are present primarily at the Golgi apparatus in premature young leaves in transgenic AtsPLA₂-α:RFP plants, while these signals are detected primarily in the apoplasts in mature leaves (Jung et al. 2012). Also, translocation of AtsPLA₂-α to the apoplast is stimulated by bacterial infection in premature young leaves. In contrast, the signal of GFP:OssPLA₂-α merges with that of an endoplasmic marker in onion epidermal cells, which indicates that the transiently expressed OssPLA₂-α localizes to the ER (Singh et al. 2012). RFP fusion protein, but not GFP and YFP, is relatively stable in the apoplasts. Thus, it is desirable to use RFP fusion protein rather than GFP or YFP fusion proteins for apoplastic localization studies.

2.2 sPLA₂ Functions

Plant PLA₂s have been implicated in important physiological processes such as development, senescence, biotic and abiotic stress responses, and the induction of secondary metabolite accumulation (Lee et al. 2005; Mansfeld 2009; Ryu 2004; Wang 2001). Lysophosphatidylcholine (LPC) and linolenic acid, which are the products of PLA₂, induce a decline in pH and accelerate the elongation of corn coleoptiles (Yi et al. 1996). Also, the PLA₂ inhibitors aristolochic acid and manoalide inhibit the auxin-induced pH decrease and coleoptile elongation. These results suggest that PLA₂ is activated by auxin, and its products induce acidification of the apoplast by activating the H⁺ pump through the signal transduction pathway of protein kinase, which in turn promotes corn coleoptile elongation (Yi et al. 1996). This hypothesis was supported by Lee et al. (2003) using transgenic plants. These authors reported that AtsPLA ₂ -β transcripts are induced by auxin treatment. In addition, RNA interference-mediated silencing (RNAi) of AtsPLA ₂ -β expression retards cell elongation, while overexpression of AtsPLA ₂ -β promotes cell elongation. Moreover, AtsPLA ₂ -β overexpressors exhibit faster stomatal opening than wild type, and AtsPLA₂-β-RNAi plants exhibit delayed light-induced stomatal opening, which can be rescued by exogenous application of LPC or lysophosphatidylethanolamine (LPE). Also, exogenous applications of LPC or LPE enhance stomatal opening in wild-type plants (Seo et al. 2008). In contrast, AtsPLA₂-β, AtsPLA₂-γ, and AtsPLA₂-δ are involved in pollen development and pollen germination (Kim et al. 2011b). AtsPLA ₂ -β may play a more vital role in pollen development, while AtsPLA ₂ -γ and -δ may function in pollen germination and pollen tube growth. Also, pollen germination is inhibited by the application of sPLA₂ inhibitors and is recovered by exogenous application of LPE but not of LPC or lysophosphatidic acid (LPA). These results indicate that LPE in particular is a key signal molecule in pollen germination and tube growth (Kim et al. 2011b).

Both AtsPLA₂-α-RNAi Arabidopsis plants and Arabidopsis seedlings treated with the sPLA₂ inhibitor ONO-RS-082 exhibit significantly disrupted plasma membrane (PM) localization of PINs in the root tissues, causing internal PIN compartments to form (Lee et al. 2010). Application of exogenous LPE restores the PM localization of PINs in an AtsPLA ₂ -α mutant and in ONO-RS-082-treated seedling. These results indicate that AtsPLA₂-α modulates PIN-FORMED protein trafficking to the PM in Arabidopsis roots, thereby revealing that sPLA₂ also plays an important role in intracellular membrane trafficking in plants (Lee et al. 2010). AtsPLA₂-α was also suggested to be as a negative regulator of the defense response through its interaction with AtMYB30, a transcription factor that functions in the hypersensitive response in short-day conditions (Froidure et al. 2010). Intriguingly, the regulation does not depend on the enzymatic activity of AtsPLA₂-α but on the just physical binding of AtsPLA₂-α to A+MYB30.

Aristolochic acid reduces root elongation and causes radial swelling of the root tip caused by microtubule disorganization (Gardiner et al. 2008). This indicates that PLA₂ is involved in microtubule organization and anisotropic growth. Another study employing aristolochic acid suggests that PLA₂ plays a critical role in programmed cell death induced by misexpression of fatty acid elongation, likely involving the exchange of very long chain fatty acids (VLCFAs) between phospholipids and the acyl-CoA pool (Reina-Pinto et al. 2009). PLA₂ is involved in salt stress-induced LPA production in the unicellular green alga Chlamydomonas (Meijer et al. 2001). LPA accumulates in Chlamydomonas under conditions of salt and nonionic hyperosmotic stress. The fact that LPA is generated by PLA₂ was recently confirmed using differential ³²P-radiolabeling experiments (Arisz and Munnik 2011).

3 Phospholipase A1

Plant PLA₁s are classified based on the presence of N terminal stretches, sequence similarities in the catalytic region, and substrate specificity. These PLA₁s include group I, II, and III PLA₁, phosphatidic acid-specific PLA₁ (PA-PLA₁), and lecithin:cholesterol acyltransferase-like PLA₁ (LCAT-PLA₁) in Arabidopsis and group I and II PLA₁ and PA-PLA₁ in rice (Chen et al. 2011; Singh et al. 2012).

3.1 Class I PLA₁s

Class I PLA₁s include seven PLA₁ genes in Arabidopsis and eight in rice. Class I PLA₁s are defined by the presence of a putative N terminal chloroplast-targeting signal (Chen et al. 2011; Seo et al. 2009). All class I PLA₁s have a GXSXG motif in the lipase class 3 domain and catalytic triad (Ser, Asp and His residues). All GFP-class I PLA₁ fusion proteins localize to the chloroplast (Ellinger et al. 2010; Grienenberger et al. 2010; Hyun et al. 2008; Ishiguro et al. 2001; Seo et al. 2009). However, a second opinion about the subcellular localization of AtPLA₁-Iα1 has recently emerged (Ellinger et al. 2010). In this study, AtPLA₁-Iα1 was co-localized with cytoplasmic lipid bodies instead of localizing to chloroplasts.

Defective in Anther Dehiscence1 (DAD1, AtPLA ₁ -Iβ1) was the first reported PLA₁ enzyme in Arabidopsis. This enzyme preferentially hydrolyzes phosphatidylcholine (PC) at the sn-1 position and regulates anther dehiscence in flowers by releasing linolenic acid for the initial step of JA biosynthesis (Ishiguro et al. 2001). DONGLE (DGL, AtPLA ₁ -Iα1) and DAD1 are necessary and sufficient for JA production (Hyun et al. 2008). DGL plays a specific role in maintaining basal JA content under normal conditions. During wounding, DGL was shown to be required for the rapid JA burst in the early phase, and DAD1 was shown to play a role in the late phase of JA production. However, the initial step in the biosynthesis of wound- and pathogen-induced JA production remains controversial. Ellinger et al. (2010) suggest that AtPLA ₁ -Iγ1 is a novel target gene for the manipulation of jasmonate biosynthesis and that, in addition to DAD1 and AtPLA ₁ -Iγ1, as yet unidentified enzymes with sn-1 and sn-2 hydrolase activity are involved in stress-induced JA formation, indicating that there is functional redundancy within the lipase family.

3.2 Class II PLA₁s

Class II PLA₁s comprise four PLA₁ genes in Arabidopsis and three in rice (Chen et al. 2011; Singh et al. 2012). Based on their sequence homology, PLA₁s from other species are also included in class II, including Dclipase from Dianthus caryophyllus (carnation) (Hong et al. 2000), LeLID1 from Lycopersicon esculentum (tomato) (Matsui et al. 2004) and CaPLA ₁ from Capsicum annuum (hot pepper) (Seo et al. 2007). Class II PLA₁s lack N terminal signal peptides and are predicted to localize to the cytosol. Cytosolic localization of AtPLA₁-IIδ and DAD1-like Seedling Establishment-related Lipase (AtDSEL, AtPLA₁-IIγ) were confirmed through GFP fusion protein studies (Kim et al. 2011a; Lo et al. 2004). AtPLA ₁ -IIδ expression is induced by treatment with sublethal levels of UV-B. Plants with suppressed AtPLA ₁ -IIδ expression via antisense technology exhibit increased tolerance to sublethal levels of UV-B stress and are unable to upregulate the expression of pathogenesis-related protein 1 (PR-1) in response to UV-B treatment. These results indicate that AtPLA₁-IIδ is capable of deesterifying membrane phospholipids and is induced in response to UV-B irradiation (Lo et al. 2004). Recombinant AtDSEL expressed in Escherichia coli (E. coli) shows a preference for 1,3-diacylglycerol and 1-monoacylglycerol over PC, which suggests that AtDSEL is a sn-1-specific lipase.

AtDSEL overexpressors are defective in post-germinative seedling growth on medium lacking an exogenous carbon source; this phenotype is rescued by the addition of sucrose to the growth medium. By contrast, atdsel-1 and atdsel-2 exhibit a mildly fast-growing phenotype in the absence of an exogenous carbon source. AtDSEL-overexpressors retained numerous peroxisomes and oil bodies in their 5-day-old cotyledons, while these organelles are exhausted in wild-type or mutant cotyledons. These results suggest that AtDSEL is involved in the negative regulation of seedling establishment by inhibiting the breakdown of storage oils (Kim et al. 2011a). Dclipase transcript levels increase just as carnation flowers begin to senesce, and the expression of this gene is also induced by ethylene treatment. Southern blot analysis confirmed that these flowers contain a single copy of Dclipase. Dclipase is predicted to be involved in mediating the onset of senescence (Hong et al. 2000). Recombinant LeLID1 protein exhibits high activity against triacylglycerols (TAGs) with long acyl chains but little activity against PC or monogalactosyldiacylglycerol. Transcript levels of LeLID1 increase rapidly in seeds during germination, reaching a maximum level just before cotyledon opening, followed by a rapid decrease. Low levels of LeLID1 expression are detected in flowers and fruits, while none can be detected in roots. LeLID1 is thought to function as a lipase during lipid mobilization to liberate fatty acids from TAG’s stored in oil bodies (Matsui et al. 2004).

3.3 Class III PLA₁

There is only one class III PLA₁ in the Arabidopsis genome and none in the rice genome (Chen et al. 2011; Singh et al. 2012). Recombinant DAD1-like acylhydrolase (AtDLAH, AtPLA₁-III) displays a stronger preference for 1-lysophosphatidylcholine, 1-monoacylglycerol, and phosphatidic acid than for PC, which indicates that AtDLAH is an sn-1-specific acylhydrolase. AtDLAH exclusively localizes to the mitochondria. Seeds of Arabidopsis AtDLAH overexpressors are more tolerant to accelerated-aging treatment than wild type, and thus these seeds have higher germination rate than wild-type seeds. By contrast, atdlah knockout mutant seeds are susceptible to accelerated-aging conditions. These results suggest that AtDLAH plays an important role in Arabidopsis seed viability (Seo et al. 2011).

4 PA-PLA₁

SGR2 in Arabidopsis is classified as a PA-PLA₁ based on the similarity of its domain structures to that of a mammalian PA-PLA₁. One gene of PA-PLA₁ was found to be present in the Arabidopsis and rice genome. The fusion protein of AtPA-PLA₁ and GFP localizes to the membranes of vacuoles and small organelles. However, the localization of OsPA-PLA₁ was predicted to be nuclear using in silico tools. AtPA-PLA₁ mutants exhibit abnormal gravitropism in inflorescence stems and hypocotyls and irregularly shaped seeds. Mutant analysis suggests that AtPA-PLA₁ is involved in a vacuolar membrane system that affects the early step of shoot gravitropism. Microarray data and quantitative RT-PCR in rice show that OsPA-PLA ₁ transcripts are upregulated in response to salt and drought stress (Kato et al. 2002; Morita et al. 2002; Singh et al. 2012). In animals, LPA is a phospholipid mediator with multiple biological roles, functions via interactions with G protein-coupled seven-transmembrane receptors (GPCRs), and is implicated in various human diseases. While two pathways for LPA production have been identified in animal cells, little is known about the function of LPA at the molecular level. It was demonstrated that LPA produced by hair follicle-specific PA-PLA₁ is an important signaling molecule for hair follicle development, which functions by modulating epidermal growth factor receptor signaling (Aoki et al. 2008; Chen et al. 2011; Inoue et al. 2011). However, PLA₁ activity was not detected with recombinant AtPA-PLA₁ proteins expressed in E. coli. Moreover, GPCRs are not found in plants. Elucidating the mechanisms of PA-PLA₁ function in plants will be challenging.

5 LCAT-PLA1

During a search for plant genes encoding enzymes involved in sterol esterification by free acids, At3g03310 was identified as lecithin:cholesterol acyltransferase (LCAT) (Noiriel et al. 2004). This gene has sequence homology with the recently discovered gene encoding phosphatidylcholine:diacylglycerol acyltransferase from Saccharomyces cerevisiae (ScPDAT). PLA₁ activity was first revealed by LCAT or PDAT assays, and the gene encoding PLA₁ was designated AtLCAT-PLA ₁ (Chen et al. 2011; Noiriel et al. 2004). Yeast expressing AtLCAT-PLA ₁ accumulates TAG (Noiriel et al. 2004). Thus, AtLCAT-PLA₁ may play a role in acyl-editing during TAG formation (Bates et al. 2009; Lu et al. 2009) but not in signaling.

6 Conclusions

Recent discoveries have significantly advanced our understanding of the biochemical and genetic requirements of distinct phospholipid signaling in plants. However, several unanswered questions still remain. For example, which isoforms of the PLA families are activated in response to specific external stimuli? What are the downstream targets of the lipid signals that are generated by PLA? These targets may be lysophospholipid-specific receptors, protein kinases, mitogen-activated protein kinase, or other signaling enzymes. And what are the upstream regulators of PLA activation? These regulators may take the form of receptors, activators, inhibitors, or hormones. The next coming years will likely produce significant advances towards the elucidation of PLA-mediated membrane phospholipid signaling in plants at both the biochemical and genetic levels. Moreover, the construction of knockout mutants and activation tagging lines, as well as the analysis of rapid protein activation without involving gene expression, will help clarify the phospholipid-derived signaling cascades.