Overexpression of patatin-related phospholipase AIIIβ altered the content and composition of sphingolipids in Arabidopsis

In plants, fatty acids are primarily synthesized in plastids and then transported to the endoplasmic reticulum (ER) for synthesis of most of the complex membrane lipids, including glycerolipids and sphingolipids. The first step of sphingolipid synthesis, which uses a fatty acid and a serine as substrates, is critical for sphingolipid homeostasis; its disruption leads to an altered plant growth. Phospholipase As have been implicated in the trafficking of fatty acids from plastids to the ER. Previously, we found that overexpression of a patatin-related phospholipase, pPLAIIIβ, resulted in a smaller plant size and altered anisotropic cell expansion. Here, we determined the content and composition of sphingolipids in pPLAIIIβ-knockout and overexpression plants (pPLAIIIβ-KO and -OE). 3-keto-sphinganine, the product of the first step of sphingolipid synthesis, had a 26% decrease in leaves of pPLAIIIβ-KO while a 52% increase in pPLAIIIβ-OE compared to wild type (WT). The levels of free long-chain base species, dihydroxy-C18:0 and trihydroxy-18:0 (d18:0 and t18:0), were 38 and 97% higher, respectively, in pPLAIIIβ-OE than in WT. The level of complex sphingolipids ceramide d18:0–16:0 and t18:1–16:0 had a twofold increase in pPLAIIIβ-OE. The level of hydroxy ceramide d18:0–h16:0 was 72% higher in pPLAIIIβ-OE compared to WT. The levels of several species of glucosylceramide and glycosylinositolphosphoceramide tended to be higher in pPLAIIIβ-OE than in WT. The total content of the complex sphingolipids showed a slightly higher in pPLAIIIβ-OE than in WT. These results revealed an involvement of phospholipase-mediated lipid homeostasis in plant growth.


INTRODUCTION
Lipids are structural components of membrane bilayers and play important metabolic and regulatory roles in plant growth, development, and stress responses. Phospholipases are major enzyme families that catalyze many of the reactions in lipid metabolism and signaling. Recently, multiple biological functions have been revealed for patatin-related phospholipase As (pPLAs; Li and Wang, 2014). Patatin-related PLAs in Arabidopsis comprise pPLAI, pPLAII (α,β,γ,δ,ε), and pPLAIII (α,β,γ,δ;Scherer et al., 2010). pPLAI has a positive role in plant resistance to the fungus pathogen Botrytis cinerea, possibly by mediating the production of jasmonates (Yang et al., 2007). Deficiency of pPLAIIα decreases resistance to bacterial pathogens and impedes oxylipin production under drought stress (La Camera et al., 2005;Yang et al., 2012). pPLAIIγ, pPLAIIδ, and pPLAIIε are involved in the response to phosphorus deficiency and auxin treatment in terms of root elongation (Rietz et al., 2004(Rietz et al., , 2010. pPLAIIIs possess a distinctive non-canonical esterase motif GxGxG, instead of GxSxG, which is present in pPLAI and pPLAIIs (Scherer et al., 2010). Overexpression of pPLAIIIδ leads to a stunted plant statue (Huang et al., 2001). Overexpression of pPLAIIIβ results in smaller plant size and reduced cellulose content in stems (Li et al., 2011). Disruption of rice DEP3, a homolog of pPLAIIIδ, results in taller rice plants (Qiao et al., 2011). Heterogeneous overexpression of an Oncidium OSAG78, another homolog of pPLAIIIδ, results in a smaller plant size and a delayed flowering time in Arabidopsis (Lin et al., 2011). These lines of evidence indicate pPLAIIIs are important for plant growth and development.
Maintenance of sphingolipid homeostasis is critical for plant growth and development (Chen et al., 2006;Dietrich et al., 2008;Teng et al., 2008;Kimberlin et al., 2013). T-DNA disruption of LCB1 gene in Arabidopsis results in an arrested development of the embryo at the globular stage (Chen et al., 2006). Partial RNA interference suppression of LCB1 results in reduced cell expansion, a smaller plant, and elevated levels of saturated sphingolipid LCBs (Chen et al., 2006). There is no apparent growth phenotype for mutants deficient in either LCB2a or LCB2b, however, the deficiency of both is lethal for gametophyte (Dietrich et al., 2008). Inducible suppression of LCB2b results in cell necrosis and reduced levels of LCBs in adult Arabidopsis plants (Dietrich et al., 2008).
The function of SPT can be regulated by small polypeptides designated as small subunits of SPT (ssSPT). ssSPTa and ssSPTb interact with SPT and stimulate its activity in Arabidopsis (Kimberlin et al., 2013). T-DNA disruption of ssSPTa results in reduced plant growth and pollen lethality in Arabidopsis (Kimberlin et al., 2013). Overexpression of ssSPTa leads to increased levels of free LCBs and LCBPs compared with that of WT, while RNA interference suppression of ssSPTa has opposite effects (Kimberlin et al., 2013). Overexpression of ssSPTa results in a greater reduction in plant growth than suppression does, when plants are treated by fumonisin B1, an inhibitor of sphingolipid synthesis (Kimberlin et al., 2013).
Previously, we reported that overexpression of pPLAIIIβ results in a reduced plant growth in Arabidopsis (Li et al., 2011). Here we report the effects of overexpression of pPLAIIIβ on the content and composition of sphingolipids, including free sphingolipids and complex ones. Our results show that overexpression of pPLAIIIβ results in an elevated level of 3-keto-sphingaine (3-KS), the product of the first step of sphingolipid synthesis, as well as altered levels of many of the species of complex sphingolipids.
The level of 3-KS was 26% lower in β-KO and 52% higher in β-OE compared with that of WT (Figure 2A). The levels of LCB t18:0 and t18:1 were approximately 15 times higher than LCB d18:0 and d18:1 in leaves of WT ( Figure 2B). Among the LCB species, the levels of d18:0 and t18:0 were 38 and 97% higher, respectively, in leaves of β-OE compared to those of WT ( Figure 2B). The level of LCB t18:0 tended to be lower in β-KO than in WT ( Figure 2B). Of the LCBP species, the level of t18:0 tended to be 85% higher while it was 43% lower in β-KO than in WT ( Figure 2C).
The fatty acyl chains of Cer can be hydroxylated to produce hydroxyl ceramide (hCer; Figure 1B). For example, the hydroxylation of 16:0 in Cer d18:0-16:0 led to the formation of hCer d18:0-h16:0 ( Figure 1C). The levels of hCer species containing one of the four types of LCBs and one the 14 types of hydroxylated fatty acyl chains were profiled (Figure 4). The most profound alteration was the level of hCer d18:0-h16:0; it was 24% lower in β-KO and 72% higher in β-OE than in WT (Figure 4). The levels of the other hCer species did not display any significant alteration between WT and β-OE (Figure 4).
pPLAIIIβ and pPLAIIIδ can hydrolyze PC and generate LPC and FA (Li et al., 2011(Li et al., , 2013a. It is implicated that pPLAIIIs are involved in the fatty acyl trafficking from plastids to ER (Li et al., 2013a). Overexpression of pPLAIIIβ may promote the fatty acyl flux from plastids to ER and enlarge certain fatty acyl pools that provide fatty acyl substrates for sphingolipid synthesis. We observed that the level of 3-KS, the precursor of sphingolipid synthesis, was significantly increased in pPLAIIIβ-overexpression plants. The alteration of this critical first step of sphingolipid synthesis could lead to the observed changes in sphingolipid homeostasis (Figure 8).
Sphingolipids are the major components of the plasma membrane (Sperling et al., 2004). Changes in sphingolipid homeostasis may alter structure integrity of raft-like domains in the plasma membrane and therefore influence cell surface activities, such as lipid trafficking and cell wall metabolism (Mongrand et al., 2004;Borner et al., 2005;Melser et al., 2011). Overexpression of pPLAIIIβ results in a decreased level of cellulose content,   a loss of anisotropic cell expansion, and a thinner cell wall (Li et al., 2011). Plasma membrane dynamics contribute significantly to the buildup of the cell wall (Li et al., 2013b). It could be possible that the altered sphingolipid homeostasis in pPLAIIIβ mutants impairs cell membrane activities which consequently results in a reduced cellulose production and plant growth.

Frontiers in Plant Science | Plant Physiology
Multiple lines of evidence suggest that pPLAIIIβ plays a role in auxin transport. In the early seedling stage, some auxin-related phenotypes were shown for pPLAIIIβ mutants, such as slightly longer roots and hypocotyls in pPLAIIIβ-KO mutants and much shorter roots and hypocotyls, as well as smaller leaves in pPLAIIIβ-OE (Li et al., 2011). Reduced lobe formation in the interdigitating pattern of leaf epidermis cells in pPLAIIIβ-OE resembles those observed in auxin receptor mutant abp1 (auxin-binding protein1; Xu et al., 2011). In addition, the induction of early auxin response genes was delayed in pPLAIIIβ-KO mutants (Labusch et al., 2013).
The altered sphingolipid composition in pPLAIIIβ mutants may disturb the auxin transport. Alteration of pPLAIIIβ expression changed the levels of sphingolipid metabolites, particularly species with saturated long chain base and saturated fatty acyl chain, such as Cer d18:0-16:0 and t18:0-16:0, hCer d18:0-h16:0, Frontiers in Plant Science | Plant Physiology FIGURE 8 | Proposed role of pPLAIIIβ on sphingolipid synthesis. In plants, fatty acids are primarily synthesized in plastids and need to be transferred to the ER for assembly of glycerolipids, such as PC and PE, as well as sphingolipids, such as long chain bases(LCBs), Cer, hCer, and GlcCer (Bates et al., 2013;Markham et al., 2013). The synthesis of GIPC takes place at Golgi apparatus ). An acyl flux cycle was proposed for the trafficking of fatty acids from plastids to ER, in which the synthesis of PC was catalyzed by LPCAT and the hydrolysis of PC by PLAs (Lands, 1960;Wang et al., 2012). pPLAIIIβ could be one type of PLA that participates in the acyl flux cycle and contributes to synthesis of the complex membrane lipids. 3-KS, 3-keto-sphinganine; Cer, ceramide; ER, endoplasmic reticulum; FA, free fatty acids; GlcCer, glucosylceramide; GIPC, glycosylinositolphosphoceramide; hCer, hydroxyceramide; LPC, lysophosphatidylcholine; LPCAT, LPC acyltransferase; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PLA, phospholipase A; SPT, serine-palmitoyltransferase. and GIPC d18:0-h16:0, and t18:0-h16:0 (Figures 3-6). Disruption of CS genes diminished the production of sphingolipids with very long chain fatty acids (>18C), impaired the auxin transport, and led to auxin defective phenotypes (Markham et al., 2011). Important functions of sphingolipids on the trafficking of auxin carriers PIN1 (PIN-Formed 1) and AUX1 (Auxin Resistant 1) were evidenced by detailed analyses of an auxin transporter, ATP-binding cassette B19 (ABCB19) auxin transporter (Yang et al., 2013). Sphingolipids are essential components of membrane microdomains or lipid rafts where they attract a unique subset of proteins and together are transported to the plasma membrane (Klemm et al., 2009). The presence of very long chain fatty acids and saturated long carbon chains in sphingolipids can increase their hydrophobicity and the transition from a fluid to a gel phase, which are required for microdomain or lipid raft formation. The altered levels of sphingolipids with saturated acyl chains in pPLAIIIβ mutants may impact the membrane physical properties, the membrane functions on auxin transport, the induction of auxin response gene expression, and subsequently the auxin-related growth.
In summary, our data show that overexpression of pPLAIIIβ alters sphingolipid homeostasis. Our study implies that pPLAIIIβ may influence the substrate availability of the first step of sphingolipid synthesis, which may alter the sphingolipid homeostasis, change the membrane integrity, and eventually impede plant growth.

PLANT GROWTH CONDITION AND GENERATION OF OVEREXPRESSION MUTANTS
Plants were grown in growth chambers with a 12 h light/12 h-dark cycle, at 23/21 • C, in 50% humidity, under 200 μmol m −2 sec −1 of light intensity, and watered with fertilizer once a week. The WT and the mutant Arabidopsis are in Columbia-0 background (Col-0). To overexpress pPLAIIIβ, the genomic sequence of pPLAIIIβ was obtained by PCR using Col-0 Arabidopsis genomic DNA as a template. The genomic DNA was cloned into the pMDC83 vector before the GFP-His coding sequence. The expression was under the control of the 35S cauliflower mosaic virus promoter. The detailed procedure to generate overexpression lines of pPLAIIIβ was described previously (Li et al., 2011).

SPHINGOLIPID PROFILING
Leaves from 4 week old plants were harvested and immediately immersed into liquid nitrogen. The frozen samples were lyophilized and stored at −80 • C before sphingolipid extraction. Approximately 30 mg of freeze-dried Arabidopsis leaves was processed for the sphingolipid profiling using mass spectrometry. The detailed protocols of sphingolipid extraction, detection, and quantification were described previously (Markham and Jaworski, 2007;Markham, 2013).