DIACYLGLYCEROL KINASE 5 regulates polar tip growth of tobacco pollen tubes

Abstract

Pollen tubes require a tightly regulated pectin secretion machinery to sustain the cell wall plasticity required for polar tip growth. Involved in this regulation at the apical plasma membrane are proteins and signaling molecules, including phosphoinositides and phosphatidic acid (PA). However, the contribution of diacylglycerol kinases (DGKs) is not clear.We transiently expressed tobacco DGKs in pollen tubes to identify a plasma membrane (PM)-localized isoform, and then to study its effect on pollen tube growth, pectin secretion and lipid signaling. In order to potentially downregulate DGK5 function, we overexpressed an inactive variant.Only one of eight DGKs displayed a confined localization at the apical PM. We could demonstrate its enzymatic activity and that a kinase-dead variant was inactive. Overexpression of either variant led to differential perturbations including misregulation of pectin secretion. One mode of regulation could be that DGK5-formed PA regulates phosphatidylinositol 4-phosphate 5-kinases, as overexpression of the inactive DGK5 variant not only led to a reduction of PA but also of phosphatidylinositol 4,5-bisphosphate levels and suppressed related growth phenotypes.We conclude that DGK5 is an additional player of polar tip growth that regulates pectin secretion probably in a common pathway with PI4P 5-kinases.

DIACYLGLYCEROL KINASE 5 participates in flagellin-induced signaling in Arabidopsis

“…Since the DGK5 sequence did not contain any known phospholipid-binding domains or polybasic amino acid stretches, a 3D homology model of DGK5 was constructed and analyzed for putative membrane-binding regions. This suggested that DGK5 had a compact, globular, heart-shaped structure, corresponding to predictions for clade III DGK isoforms of tobacco ( Scholz et al, 2022 ). The mapping of surface electrostatic potential onto the model revealed that DGK5 had a strong bipolar charge distribution with a negatively charged pocket surrounded by an area with positive surface charge ( Figure 7C ) that could be responsible for targeting to negatively charged PM lipids.…”

“…In A. thaliana , the DGK gene family contains seven members belonging to three clusters according to their domain structures and sequence similarities ( Gómez-Merino et al, 2004 ; Escobar-Sepúlveda et al, 2017 ; Scholz et al, 2022 ). To identify the DGK isoforms contributing to flg22-triggered PA production, a screening of different knock-out mutant lines dgk1 , dgk4 , and dgk5.1 that included members of all three DGK clusters was carried out.…”

“…To this end, the conserved Gly112 in the catalytic domain, which is important for enzymatic activity ( Franks et al, 2017 ), was replaced by an Ala (G112A). The same strategy was recently used in plants for the DGK5 ortholog from tobacco ( Nicotiana tabacum ) NtDGK5 ( Supplemental Figure S8 and Scholz et al, 2022 ). The level of DGK5 transcripts was measured in seedlings of all transgenic lines by RT-qPCR and the presence of DGK5-GFP protein in all GFP-tagged lines was verified using anti-GFP antibodies ( Supplemental Figure S9 ).…”

“…DGK activity has been reported in several plant species including tobacco, wheat ( Triticum aestivum ), tomato, Arabidopsis, rice ( Oryza sativa ), and apple ( Malus prunifolia ) ( Escobar-Sepúlveda et al, 2017 ). Plant DGKs can be separated into three clusters ( Gómez-Merino et al, 2004 , 2005 ; Scholz et al, 2022 ). AtDGK1 and AtDGK2 are part of cluster I and they possess a hydrophobic segment required for membrane targeting ( Vaultier et al, 2008 ).…”

Comprehensive analysis of glycerolipid dynamics during tobacco pollen germination and pollen tube growth

“…Multiple reports also pinpointed the role of several DGK isoforms: in Arabidopsis, ER-localized DGK2 and DGK4 are required for gametogenesis and pollen tube growth ( Dias et al., 2019 ; Angkawijaya et al., 2020 ). Tobacco DGK5 is involved in the polar secretion of cell wall material to the pollen tube tip, possibly by regulating phosphoinositide-signaling pathways ( Scholz et al., 2022 ). Our multifaceted data on PA generation and dynamics strongly suggest that in tobacco pollen, PA production comes mainly from PC and, to a lesser extent, from PE, as can be inferred from individual species profiles.…”