A pair of nonspecific phospholipases C, NPC2 and NPC6, are involved in gametophyte development and glycerolipid metabolism in Arabidopsis

Abstract

Phospholipases play crucial roles in plant membrane lipid homeostasis. Nonspecific phospholipase C (NPCs) establish a unique class of phospholipases found only in plants and certain bacteria. Here, we show that two previously uncharacterized NPC isoforms, NPC2 and NPC6, are required for male and female gametophyte development in Arabidopsis. Double mutant plants of npc2-1 npc6-2 could not be retrieved because npc2-1 npc6-2 ovule and pollen development is affected. Genetic complementation, reciprocal crossing and microscope observation of npc2-1/- npc6-2/+ and npc2-1/+ npc6-2/- plants suggest that NPC2 and NPC6 are redundant and are required for normal gametophyte development. Both NPC2 and NPC6 proteins are localized to the plastids. Promoter-GUS assays in transgenic Arabidopsis revealed that NPC2 and NPC6 are preferentially expressed in floral organs rather than in leaves. In vitro enzyme assays showed that NPC2 and NPC6 hydrolyze phosphatidylcholine and phosphatidylethanolamine, but not phosphatidate, being consistent with the reported substrate selectivity of NPCs. The amounts of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol were increased in buds but not in flowers of npc2-1/- npc6-2/+ and npc2-1/+ npc6-2/- plants, presumably due to reduced phospholipid hydrolysis activity in developing flowers. Our results demonstrate that NPC2 and NPC6 play crucial roles in gametogenesis during flower development.

Non‐specific phospholipases C, NPC2 and NPC6, are required for root growth in Arabidopsis

“…Ovaries of knock‐down mutants showed a number of underdeveloped ovules (Figure q). Hence, the knock‐down mutants of NPC2 and NPC6 phenocopied the gametophyte developmental defect observed in npc2‐1/− npc6‐2 /+ or npc2‐1/+ npc6‐2 / − (Ngo et al ., ).…”

“…Our knock‐down mutant lines allowed for observing vegetative growth, which was not possible with gametophytic‐lethal double‐knock‐out mutants (Ngo et al ., ). Although we observed no growth or lipid phenotypes in rosette leaves, the knock‐down mutant lines showed significantly early flowering phenotype under long‐day but not short‐day conditions (Figure d).…”

“…NPC2 and NPC6 may play a redundant role in the NPC pathway, whereas PMT1 may play a more dominant role than PMT2 in the PMT pathway because the pmt1 mutant showed a shorter root length and pmt2 enhanced the root phenotype only in a pmt1 background (Cruz‐Ramírez et al ., ; Liu et al ., ). We highlight an emerging function for NPC2 and NPC6 in roots to promote growth, a distinct role from what was previously assessed in gametogenesis (Ngo et al ., ). The role of PCho in root growth remains enigmatic (Cruz‐Ramírez et al ., ; Chen et al ., ; Liu et al ., , ; Chen et al ., ).…”

“…To address a possible function of NPC2 and NPC6 beyond gametogenesis, we created multiple transgenic gene knock‐down mutant lines by using artificial microRNA (amiRNA) technology to suppress the gene expression of one isoform in the null mutant background of the others ( Pro35S:amiNPC2 in npc6‐2 and Pro35S:amiNPC6 in npc2‐1 ). These knock‐down mutant lines confirmed previously reported gametophytic phenotypes in npc2‐1/+ npc6‐2/− and npc2‐1/− npc6‐2 /+ (Ngo et al ., ). No morphological or lipid compositional phenotypes were observed in leaves, but roots were shorter with an extra cell division in columella cells.…”

“…In Arabidopsis, six isoforms of NPC are known (Nakamura et al, 2005) and were found to be involved in the stress response under phosphate deficiency (NPC4 and NPC5: Nakamura et al, 2005;Gaude et al, 2008), salinity (NPC4 and NPC5: Peters et al, 2010Peters et al, , 2014Kocourkov a et al, 2011), heat shock (NPC1: Kr ckov a et al, 2015), and immunity (NPC2: Kr ckov a et al, 2018). Recently, we showed that NPC2 and NPC6 redundantly play an essential role in gametogenesis because double-knock-out mutants showed a gametophytic-lethal phenotype (Ngo et al, 2018). Although this finding indicates that NPC2 and NPC6 are essential in plant viability, their specific function beyond the gametogenesis remains elusive because of the lethality of the double homozygous mutant.…”