Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a low-abundance membrane lipid essential for plasma membrane function1,2. In plants, mutations in phosphatidylinositol 4-phosphate (PI4P) 5-kinases (PIP5K) suggest that PI(4,5)P2 production is involved in development, immunity and reproduction3,4,5. However, phospholipid synthesis is highly intricate6. It is thus likely that steady-state depletion of PI(4,5)P2 triggers confounding indirect effects. Furthermore, inducible tools available in plants allow PI(4,5)P2 to increase7,8,9 but not decrease, and no PIP5K inhibitors are available. Here, we introduce iDePP (inducible depletion of PI(4,5)P2 in plants), a system for the inducible and tunable depletion of PI(4,5)P2 in plants in less than three hours. Using this strategy, we confirm that PI(4,5)P2 is critical for various aspects of plant development, including root growth, root-hair elongation and organ initiation. We show that PI(4,5)P2 is required to recruit various endocytic proteins, including AP2-µ, to the plasma membrane, and thus to regulate clathrin-mediated endocytosis. Finally, we find that inducible PI(4,5)P2 perturbation impacts the dynamics of the actin cytoskeleton as well as microtubule anisotropy. Together, we propose that iDePP is a simple and efficient genetic tool to test the importance of PI(4,5)P2 in given cellular or developmental responses, and also to evaluate the importance of this lipid in protein localization.
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The Arabidopsis lines and plasmids generated in this study are available from the corresponding authors upon request and the transgenic lines are also available at NASC as NASC stocks N2110785–N2110797. Source data are provided with this paper.
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We are grateful to the SiCE group (RDP), in particular T. Gaude and V. Bayle (RDP, Lyon), and to Y. Boutté (Laboratoire de Biogenèse Membranaire), D. Van Damme (Vlaams Instituut voor Biotechnologie (VIB)), T. Stanislas (ZMBP), F. Besnard and N. Doll (RDP), for comments and discussion. We also thank former interns L. Courgeon (ENS de Lyon) and A. Bauer (ENS de Lyon), who performed preliminary experiments during the early steps of this project and helped with plant labour, and K. Grünwald. We thank J. Berger, P. Bolland and A. Lacroix from our plant facility. We acknowledge the contribution of Structure Fédérative de Recherche Biosciences (UMS3444/CNRS, US8/Inserm, ENS de Lyon, Université Claude Bernard Lyon 1) facilities: C. Lionet, E. Chatre and J. Brocard at the LBI-PLATIM-MICROSCOPY for assistance with imaging. We are also grateful to E. Russinova (VIB) for kindly providing us with ES9-17; S. Bednarek for sharing markers and for discussion and M. Uyttewaal (INRAE) for the MBD–GFP marker. We thank E. Isono (University of Konstanz) for sharing SH3P2–GFP’s transgenic line with us. We also thank R. Boisseau (OBEE department, University of Montana) for help with the statistical analysis. This work was supported by the European Research Council (no. 3363360-APPL under FP/2007–2013, Y.J.; EU-FET 828753, T.M.), the Netherlands Organisation for Scientific Research (NWO 867.15.020; 711.017.005; T.M.), Austrian Academy of Science through the Gregor Mendel Institute (Y.B.), French National Research Agency ANR (caLIPSO; ANR-18-CE13-0025-02; Y.J.), ANR JC/JC JUNIOR INVESTIGATOR GRANT (INTERPLAY; ANR-16-CE13-0021; M.-C.C., A.F.) and a SEED FUND ENS LYON-2016 (M.-C.C.).
The authors declare no competing interests.
Peer review information Nature Plants thanks Takashi Aoyama, Bo Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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a, Alignment of (i) Homo sapiens OCRL (HsOCRL or OCRL1) truncation (HsOCRL234-539) successfully used in optogenetic systems in mammalian cells, and (ii) of Drosophila melanogaster dOCRL. We were concerned that this domain, that underwent natural selection in an endotherm organism with set-point temperature of 37 °C, would be less active in Arabidopsis thaliana, a plant of temperate climate which we usually grow at 21 °C. Therefore, we decided to use the phosphatase domain of dOCRL, Drosophila melanogaster homolog, as Drosophila are ectotherm and live at ~ 20 °C. Alignment of OCRL1234-539 and dOCRL allowed us to identify dOCRL168-509 as the homolog region of OCRL1 generally used in optogenetic systems. b, Coomassie blue staining monitoring purification of recombinant dOCRL phosphatase domain (His-SUMO-dOCRL168-509-His) from Escherichia coli. We first assessed dOCRL activity, which has not been reported so far, even though in vivo data suggest a conserved PI(4,5)P2 5-phosphatase activity. We ordered a synthetic gene corresponding to dOCRL168-509 and codon optimized for expression in Arabidopsis. We cloned a recombinant dOCRL protein (His-SUMO-dOCRL168-509-His) tagged with two 6-histidine tags and a SUMO tag, making it suitable for expression in Escherichia coli and purification. We purified His-SUMO-dOCRL168-509-His using a cobalt resin-based affinity chromatography and protein concentration IN: input, lysate of bacteria induced for the expression of His-SUMO- dOCRL168-509-His; FL: flow through; W: wash; E1 to E3: elution fraction 1 to 3; C1 and C2: concentrated fractions from pulled E1, E2 and E3. C1 and C2 were obtained separately on different days. His-SUMO-dOCRL168-509-His expected molecular weight is 54.8 kD; 3 Replicates (c) Malachite green phosphatase assay on His-SUMO-dOCRL169-509-His using short chain water-soluble phosphoinositides. We subjected the purified protein fractions C1 and C2 to Malachite Green Phosphatase assays, that detects phosphate released by dephosphorylations, in presence of short chain (diC:8) water-soluble phosphoinositides. Note that we only assessed dOCRL168-509 activity toward phosphoinositides reported in plant cells. Replicate 1 correspond to C1 fraction. Replicate 2 to C2 fractions. Mock: no phosphoinositide added.
a, Schematic drawing depicting constructs used to genetically-induced PI(4,5)P2 pool modification at the PM. After dex treatment, MAP-mCh-dOCRL is anchored at the plasma membrane and dephosphorylates PI(4,5)P2, causing the PI(4,5)P2 biosensor to be released into the cytosplasm. b, Controls used in this study includes 3xmCh and a catalytic dead version of dOCRL. After dex treatment, both MAP-3xmCh and MAP-mCh-dOCRLdead are anchored at the PM, but do not affect the pool of PI(4,5)P2. The PI(4,5)P2 biosensor is found at the PM. To build the iDePP system, we had to engineer a synthetic enzyme, which consists of the isolated catalytic domain of a 5-phosphatase (which if expressed on its own would be soluble in the cytosol and likely inactive) and then target this module to the plasma membrane, for specific depletion of PI(4,5)P2). Details can be found in the Methods section.
Extended Data Fig. 3 Successful inducible expression and subcellular targeting of MAP-mCH-dOCRL and negative controls.
a, Inducible system used in this study. We performed site-directed D468G mutation in dOCRL catalytic domain, similarly to D523G mutation already described in human OCRL1 that abolishes its phosphatase activity. We could therefore use MAP-mCH-dOCRL168-509D468G (hereafter named MAP-mCH-dOCRLdead) as negative control, together with a MAP-3xmCH recombinant protein. UBQ10 promoter is responsible for mild/strong and ubiquitous expression of GVG (GAL4 VP16 GR) synthetic gene. GR domain binds dexamethasone (dex) and subsequently induces GVG homodimerization and nuclear import. There, GAL4 domain binds UAS DNA elements and VP16 strongly activates the expression of downstream gene: MAP-mCH-dOCRL168-509, or MAP-mCH-dOCRL168-509D468G (MAP-mCH-dOCRLdead), or MAP-3xmCH. MAP is myristoylation and palmytoylation sequence, responsible for plasma membrane targeting. The mCH corresponds to monomeric CHERRY fluorescent protein. dOCRLD468G168-509 is an inactive phosphatase domain (later on called dOCRLdead). 3xmCH correspond to three fused mCHERRY. b, We addressed the timing of expression and the localization of each construct in Arabidopsis lines stably transformed with UBQ10pro:GVG:MAP-mCH-dOCRL, or UBQ10pro:GVG:MAP-mCH-dOCRLdead, or UBQ10pro:GVG:MAP-3xmCH. In root meristematic epidermal cells, without dex treatment, none of the recombinant-proteins were detected using confocal microscopy. A 16 h treatment with 0.5 µM dex led to the detection of mCH fluorescence, indicating that induction of the genetic construct had occurred. However, we observed a mosaic induction and a significant number of roots had no cells expressing the fluorescent reporter to detectable levels. To overcome these issues, we optimized the treatment to a 16 h induction with 5 µM dex. Using this set up, we robustly observed red fluorescence using confocal microscopy where MAP-mCH-dOCRL, MAP-mCH-dOCRLdead and MAP-3xmCH. Each of the synthetic protein was efficiently targeted to membranes, including the plasma membrane, where PI(4,5)P2 accumulates, and intracellular compartments. Therefore, a 16 h 5 µM dex treatment is sufficient for an effective expression of dOCRL in Arabidopsis stable transgenic lines. For all three inducible constructs we monitored mCH fluorescence without dex treatment or after a 16h-treatment with either 0.5 µM or 5 µM dex. Signal intensity is color-coded (green fire blue scale); 2 Replicates. Scale bars, 20 µm.
The 32P-PIP levels in iDePP lines and controls ± dex of 3 independent replicates. a-c, Seedlings were labelled with 32Pi and incubated ± dex O/N (16–20 hrs). Each sample contained the lipid extract of three seedlings, of which 1/5th was analyzed by TLC and quantified by phosphoimaging, of which 32P-PIP2 (a), PIP (b) and PA (c) were calculated as percentage of total 32P-lipids. d-f, Time-course analysis of 32P-PIP2 (d), PIP (e) and PA (f) levels in iDePP seedlings ± dex in MAP-mCH-dOCRL line. Seedlings of MAP-mCH-dOCRL line were labelled for 20 hrs with 32Pi and co-incubated with or without dex for the times indicated (0–20 hrs). Each sample contained the lipid extract of three seedlings of which 1/5th was analyzed by TLC and quantified by phosphoimaging. First, the mean of control on all time points was calculated. The graphs represent the calculated mean of the sample reported to the mean of the control. In the plots, middle horizontal bars represent the median, while the bottom and top of each box represent the 25th and 75th percentiles, respectively. At most, the whiskers extend to 1.5 times the interquartile range, excluding data beyond. For range of value under 1,5 IQR, whiskers represent the range of maximum and minimum values. All statistical tests were two-sided.
Extended Data Fig. 5 Graphic representation of the fluorescent intensity observed for the different biosensors, used for the quantification of the dissociation index.
Orange lines represent the means. In the plots, middle horizontal bars represent the median, while the bottom and top of each box represent the 25th and 75th percentiles, respectively. At most, the whiskers extend to 1.5 times the interquartile range, excluding data beyond. For range of value under 1,5 IQR, whiskers represent the range of maximum and minimum values.
a-c, Subcellular localization of mCIT-2xPHPLC and either MAP-mCH-dOCRL (a), or MAP-3xmCH; N = 12 (b), MAP-mCH-dOCRLdead; N = 12 (c), in cotyledon epidermal cells; N = 6. Fluorescence intensity is color-coded (green fire blue scale), cytoplasmic strands are indicated by the arrows. Scale bars, 10 µm. d-g, Induction of the iDePP system in the shoot apical meristem of Arabidopsis; N = 15 (Scale bar, 20 μm in (a-b), 10 μm in (c-d). Asterisks represent the position of the nucleus in the cell. (h) and (i) Subcellular localization of MAP-mCH-dOCRL (left panel) and mCIT-2xPHPLC (right panel) in a naked shoot meristem from an NPA-treated seedling, as viewed from the top; N = 4. In the absence of dex induction, mCIT-2xPHPLC was localized at the plasma membrane throughout the meristem, including both the central and peripheral zones (h, right panel). By contrast, upon induction of MAP-mCH-dOCRL expression, mCIT-2xPHPLC was no longer sharply localizing at the cell edge (i, right panel). Instead, mCIT-2xPHPLC accumulated in the cytosol, while still being excluded from the round central nuclei (i, right panel). Note that after dex induction, MAP-mCH-dOCRL was strongly expressed in the peripheral zone, and was only weekly expressed in the central zone (i, left panel). However, although its expression was weak in the central zone it appeared to be sufficient to impact mCIT-2xPHPLC localization. Scale bar, 20 μm. (j) and (k) Effect of PI(4,5)P2 depletion on organ initiation in NPA-treated meristems; N = 8. Altogether, these observations suggest that PI(4,5)P2 depletion seems to affect organ initiation in NPA-treated meristems. Scale bars, 700 µm.
Quantification over time (at 3, 5- and 7-days post germination) of the root length (cm) in transgenic lines MAP-mCH-dOCRL (a) and MAP-mCH-dOCRLdead (b) grown on MS ½ plate with 5 µM of dex. Details for the statistical analysis can be found in the Methods. In the plots, middle horizontal bars represent the median, while the bottom and top of each box represent the 25th and 75th percentiles, respectively. At most, the whiskers extend to 1.5 times the interquartile range, excluding data beyond. For range of value under 1,5 IQR, whiskers represent the range of maximum and minimum values.
a,b, Representative confocal images of Arabidopsis root cells coexpressing mCIT-P4MSidM with either MAP-mCH-dOCRL (a) or MAP-3xmCH (b). c,d, Confocal imaging showing the effect of the expression of MP-mCH-dOCRL on CLC2-GFP localization. e, Quantification of the effect of MAP-mCH-dOCRL expression on CLC2-GFP localization. The dissociation index is the ratio of (i) plasma membrane to cytosol fluorescence ratio without dex treatment, (ii) plasma membrane to cytosol fluorescence ratio after dex treatment. Statistical analysis with LMER (Type II Wald χ2 test) and posthoc tests; 3 replicates (f-g) Confocal imaging showing the effect of the expression of MAP-mCH-dOCRL on SH3P2-sGFP line (h) Quantification of the effect of MAP-mCH-dOCRL expression on SH3P2-sGFP localization. Per root, cells showing a delocalization of SH3P2-sGFP were manually counted for untreated and dex treated plants using FIJI ‘multi-point’ tool and compared by a generalized linear model, Poisson family; 3 replicates. In the plots, middle horizontal bars represent the median, while the bottom and top of each box represent the 25th and 75th percentiles, respectively. At most, the whiskers extend to 1.5 times the interquartile range, excluding data beyond. For range of value under 1,5 IQR, whiskers represent the range of maximum and minimum values. All statistical tests were two-sided. Scale bar, 10 μm.
Extended Data Fig. 9 Effects on the cytoskeleton of the depletion of PI(4,5)P2 using the iDePP system.
a, F-actin cytoskeleton labelled by LifeAct-YFPv in cells expressing MAP-mCH-dOCRL or MAP-3xmCH 16h after 5 µM dex; 4 replicates. b, Cortical microtubules labelled by TUA6-GFP, in non-treated cells (no dex) or treated (5 µM dex after 16 hours) to induce MAP-mCH-dOCRL or MAP-3xmCH expression; 3 replicates. All pictures are z-projections; no dex: no treatment; dex: dexamethasone treatment. Scale bars, 10 µm. c, Example of the images used for the quantification of the anisotropy using FibrilTool macro for imageJ. In the plots, middle horizontal bars represent the median, while the bottom and top of each box represent the 25th and 75th percentiles, respectively. At most, the whiskers extend to 1.5 times the interquartile range, excluding data beyond. For range of value under 1,5 IQR, whiskers represent the range of maximum and minimum values. All statistical tests were two-sided.
Time-course analysis during dex induction, monitoring mCIT-2xPHPLC PI(4,5)P2 biosensor subcellular localization at the root tip in epidermal cells every five minutes.
Time-course analysis with dex induction of MAP-mCH-dOCRL (right panel), monitoring mCIT-2xPHPLC PI(4,5)P2 biosensor subcellular localization (left panel) at the root tip in epidermal cells every five minutes.
Time-course analysis without dex induction of MAP-mCH-dOCRL (right panel), monitoring mCIT-2xPHPLC PI(4,5)P2 biosensor subcellular localization (left panel) at the root tip in epidermal cells every five minutes.
Time-lapse analysis of the root-hair growth upon induction of PI(4,5)P2 depletion using iDePP. Time-course analysis with dex induction (5 µM) of MAP-mCH-dOCRLdead (left roots) and MAP-mCH-dOCRL (right roots), monitoring root growth and root-hair formation for seven hours (images taken every second).
Time-lapse analysis of the actin dynamics upon induction of PI(4,5)P2 depletion using iDePP. Time-course analysis over two minutes, monitoring Lifeact-YFPv subcellular localization in root cells expressing MAP-mCH-dOCRL (right) or not expressing MAP-mCH-dOCRL (left). Pictures were taken every three seconds.
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Doumane, M., Lebecq, A., Colin, L. et al. Inducible depletion of PI(4,5)P2 by the synthetic iDePP system in Arabidopsis. Nat. Plants 7, 587–597 (2021). https://doi.org/10.1038/s41477-021-00907-z