Nonspecific phospholipase C4 hydrolyzes phosphosphingolipids and sustains plant root growth during phosphate deficiency

Abstract

Phosphate is a vital macronutrient for plant growth, and its availability in soil is critical for agricultural sustainability and productivity. A substantial amount of cellular phosphate is used to synthesize phospholipids for cell membranes. Here, we identify a key enzyme, nonspecific phospholipase C4 (NPC4) that is involved in phosphosphingolipid hydrolysis and remodeling in Arabidopsis during phosphate starvation. The level of glycosylinositolphosphorylceramide (GIPC), the most abundant sphingolipid in Arabidopsis thaliana, decreased upon phosphate starvation. NPC4 was highly induced by phosphate deficiency, and NPC4 knockouts in Arabidopsis decreased the loss of GIPC and impeded root growth during phosphate starvation. Enzymatic analysis showed that NPC4 hydrolyzed GIPC and displayed a higher activity toward GIPC as a substrate than toward the common glycerophospholipid phosphatidylcholine. NPC4 was associated with the plasma membrane lipid rafts in which GIPC is highly enriched. These results indicate that NPC4 uses GIPC as a substrate in planta and the NPC4-mediated sphingolipid remodeling plays a positive role in root growth in Arabidopsis response to phosphate deficiency.

Acylation of non‐specific phospholipase C4 determines its function in plant response to phosphate deficiency

“…2008; Nakamura et al, 2005;Wimalasekera et al, 2010;Yang et al, 2021). In summary, a model for the regulation of subcellular localization of NPC4 and NPC5 in plants was proposed (Figure 9).…”

“…However, the loss of NPC4 had no overt effect on decreasing PC in leaves (Nakamura et al, 2005) or in roots, except at an early stage (Su et al, 2018) during phosphate deficiency, indicating that PC may not be the major substrate of NPC4 during this stress condition. Recently, we have found that NPC4 hydrolyzes GIPC and that a loss of NPC4 in Arabidopsis impeded the decrease in GIPC induced by phosphate deficiency (Yang et al, 2021). Here, we show that introducing the non‐acylating NPC4 C533A into npc4‐1 fails to functionally restore the level of GIPC to that found in the WT, but that introducing the WT NPC4 does restore GIPC to WT level.…”

“…Samples were heated in 1 ml of tetrahydrofuran (THF)/methanol/water (2:1:2 v/v/ v) containing 0.1% formic acid and dissolved by ultrasound sonication, then centrifuged at 500 g for 10 min to remove the insoluble substance. Sphingolipids were analyzed using an Exion UPLC system coupled with a triple quadrupole/ion trap mass spectrometer (6500 Plus QTRAP; SCIEX, https://sciex.com) according to a previous method, with modifications (Yang et al, 2021).…”

“…NPC4 is membrane bound and associated with the plasma membrane (PM), whereas NPC5 is soluble and cytosolic (Andersson et al, 2005; Gaude et al, 2008). Recently, we found that NPC4 was associated with PM rafts and hydrolyzed glycosyl inositol phosphoryl ceramide (GIPC) during phosphate deprivation (Yang et al, 2021). As GIPC is enriched in PM rafts, the raft association of NPC4 may underlie a key mechanism for the function of NPC4.…”

Inositolphosphate glycans and a fucosylated xyloglucan oligosaccharide are accumulated upon Arabidopsis thaliana/ Botrytis cinerea infection

“…This suggests that a GIPC phospholipase C activity which cleaves phospholipids before the phosphate group, is specifically promoted in that condition. It is worth to note that the non-specific phospholipase C4 (NPC4) has been shown to be involved in GIPC hydrolysis in response to phosphate deficiency 20 and consequently, IPGs could act more broadly as stress-related molecules. The accumulation of serie A IG (m/z 517) also suggests that a GIPC phospholipase D, which cleaves after the phosphate moiety, could be activated.…”