Background

Glycerol-3-phosphate (G3P) is an obligatory component of energy-producing reactions including glycolysis and glycerolipid biosynthesis (Lim et al., 2017). For glycerolipid biosynthesis, G3P is first acylated with the fatty acid (FA) oleic acid (18:1), to form lyso-phosphatidic acid (lyso-PA); this reaction is catalyzed by the chloroplastic enzyme G3P acyltransferase encoded by the ACT1 gene in Arabidopsis. Lyso-PA is converted to phosphatidic acid (PA) via lysophosphatidic acid acyltransferase–catalyzed transfer of 16:0 FA to the sn-2 position of lyso-PA. PA is in turn converted to phosphatidylglycerol and diacylglycerol (DAG). The DAG pools act as precursors for the synthesis of other major plastidal membrane lipids, including monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulpholipids. This pathway of glycerolipid synthesis operates in the chloroplast and is commonly referred to as the prokaryotic pathway.

G3P levels are regulated by enzymes involved in its biosynthesis, as well as those involved in G3P catabolism (Venugopal et al., 2009). G3P is synthesized via the glycerol kinase (GK)-mediated phosphorylation of glycerol, or the G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate (DHAP) (Mandal et al., 2011; Lim et al., 2017). DHAP is derived from glycolysis via triosephosphate isomerase activity on glyceraldehyde-3-phosphate. In Arabidopsis, the total G3P pool is derived from the activities of five G3Pdh isoforms and one GK isoform. The G3Pdh enzymes are present in cytosol, mitochondria, and chloroplast, and only a mutation in the plastid-localized G3Pdh isoforms, designated GLY1, impairs glycerolipid biosynthesis (Shen et al., 2003 and 2006). Unlike GLY1, neither the second chloroplastic nor the two cytosolic isoforms of G3Pdh contribute to plastidal and/or extraplastidal lipid biosynthesis (Chanda et al., 2011). This suggests that a specific sub-pool of G3P in the chloroplast is required for lipid biosynthesis.

The cellular pool of G3P also regulates the plastidal 18:1 level, which in turn governs nitric oxide levels and thereby chloroplast-nucleus retrograde signaling associated with defense. G3P levels are also important for basal defense against the hemibiotrophic fungus, Colletotrichum higginsianum (Chanda et al., 2008). Genetic mutations affecting G3P synthesis in Arabidopsis enhance susceptibility to C. higginsianum; conversely, plants accumulating increased G3P show enhanced resistance. G3P also contributes to R-mediated defense leading to systemic acquired resistance (SAR) in Arabidopsis, soybean, and wheat (Chanda et al., 2011Yang et al., 2013; Yu et al., 2013; Gao et al., 2014; Wang et al., 2014 and 2018; Shine et al., 2019). G3P also plays an important role in root-shoot-root signaling that regulates incompatible response against rhizobia in soybean (Shine et al., 2019). Compromised SAR in G3P-deficient mutants is restored by exogenous application of G3P. G3P is systemically mobile and is transported to the distal tissues via the plasmodesmata (PD) (Lim et al., 2016). This symplastic transport of G3P involves specific isoforms of PD-localizing proteins, which regulate PD gating. Thus, quantification of G3P levels in the plant tissues is an integral part of research on plant defense. Earlier methods to quantify G3P levels from plant tissues was based on high-performance liquid chromatography–based profiling of extracts prepared from ~0.5–1 g of tissue weight. The G3P peaks in chromatograms were assigned using electrochemical detection of analytes corresponding to the G3P standard (Chanda et al., 2008 and 2011). This method was unable to confirm the precise identity of G3P; moreover, it required excessive tissue weight for reliable extraction of G3P.

In the present method, we use gas chromatography (GC)-mass spectrometry (MS)–based selective ion monitoring (SIM) mode to quantify G3P from tissues or petiole exudates. Ribitol was used as an internal standard and both ribitol and G3P were derivatized using N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) (Figure 1). This method can reliably quantify G3P from ~10–100 mg of fresh tissue, is cost effective, and can be used to process 100–200 samples in a day.

Figure 1. Derivatization reactions for analyte G3P (upper panel) and the internal standard ribitol (lower panel) with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). MSTFA reacts with hydroxyl groups and the derivatized products of G3P and ribitol contain four or five trimethylsilyl groups (TMS), respectively.