Role of Arabidopsis AtPI4Kγ3, a type II phosphoinositide 4-kinase, in abiotic stress responses and floral transition

Abstract

Phosphoinositides (PIs) are essential metabolites which are generated by various lipid kinases and rapidly respond to a variety of environmental stimuli in eukaryotes. One of the precursors of important regulatory PIs, phosphatidylinositol (PtdIn) 4-phosphate, is synthesized by PtdIns 4-kinases (PI4K). Despite its wide distribution in eukaryotes, its role in plants remains largely unknown. Here, we show that the activity of AtPI4Kγ3 gene, an Arabidopsis (Arabidopsis thaliana) type II PtdIn 4-kinase, is regulated by DNA demethylation and some abiotic stresses. AtPI4Kγ3 is targeted to the nucleus and selectively bounds to a few PtdIns. It possessed autophosphorylation activity but unexpectedly had no detectable lipid kinase activity. Overexpression of AtPI4Kγ3 revealed enhanced tolerance to high salinity or ABA along with inducible expression of a host of stress-responsive genes and an optimal accumulation of reactive oxygen species. Furthermore, overexpressed AtPI4Kγ3 augmented the salt tolerance of bzip60 mutants. The ubiquitin-like domain of AtPI4Kγ3 is demonstrated to be essential for salt stress tolerance. Besides, AtPI4Kγ3-overexpressed plants showed a late-flowering phenotype, which was caused by the regulation of some flowering pathway integrators. In all, our results indicate that AtPI4Kγ3 is necessary for reinforcement of plant response to abiotic stresses and delay of the floral transition.

Keywords: UBL domain; abiotic stress; flowering time; phosphoinositide kinase; reactive oxygen species.

Figure 1

Analyses of AtPI4Kγ3 expression under abiotic stresses. (a) Graphical display of the AtPI4Kγ genes during indicated abiotic stresses in shoot. Range of expression fold of each gene is indicated as legend (±) at right panel of respective pattern. Data from microarray analyses of the AtGenExpress abiotic stress series. (b) Expression of AtPI4Kγ3 under salt, ABA, cold, heat and drought stress. Error bars indicate the mean ±SD for each set of three independent experiments with significant difference at *P < 0.001. (c) Histochemical GUS expression pattern in 1‐week‐old proAtPI4Kγ3::GUS transgenic seedlings under control and different stress treatment as indicated. Scale bar = 2 mm.

Figure 2

Nuclear localization, protein kinase and lipid‐binding activity of AtPI4Kγ. (a) Subcellular localization of transiently expressed GFP‐AtPI4Kγ3 in tobacco epidermal cells. Images with bright field (upper left panel), DAPI channel (upper right panel), GFP channel (lower left panel) and merged (lower right panel) were observed by confocal microscope. Yellow bar within each panel represents 20 μm in length. (b) In vitro kinase assay with recombinant GST or GST‐AtPI4Kγ3 performed by autoradiography (right) and Coomassie blue staining (CBS; left). The phosphorylation reaction mixture contained [γ⁻³²P]ATP, and reactions were conducted using MPK6 (positive control) incubated with substrates as indicated. The upper two arrows indicate autophosphorylation signal of GST‐AtPI4Kγ3 and GST‐MPK6, respectively, and the lower arrow indicates phosphorylation of the substrate MBP. (c) The binding capacity of AtPI4Kγ3 to many kinds of lipids (1–15) was tested using PIP strips^™ (left panel), and the affinity of AtPI4Kγ3 for each lipids was determined using a PIP Array^™ sheet on which the indicated amounts of lipids were immobilized (right panel): 1. lysophosphatidic acid, 2. lysophosphatidylcholine, 3. phosphatidylinositol (PtdIns), 4. PtdIns(3)P, 5. PtdIns(4)P, 6. PtdIns(5)P, 7. phosphatidylethanolamine, 8. phosphatidylcholine, 9. sphingosine 1‐phosphate, 10. PtdIns(3,4)P₂, 11. PtdIns(3,5)P₂, 12. PtdIns(4,5)P_2, 13. PtdIns(3,4,5)P₃, 14. phosphatidic acid, 15. phosphatidylserine, 16. blank.

Figure 3

Overexpression of PI4Kγ3 enhances plant tolerance to high salinity. (a) Postgermination growth of WT, pi4kγ3, 35S::PI4Kγ3 #11, #20 and 35S::PI4Kγ3/pi4kγ3 #1 on MS medium with or without NaCl. Photographs were taken at 7 days after imbibition. (b) Germination rates (%) of the plants used in (a) on MS medium with indicated NaCl concentrations. Rates were scored from 1 day to 5 days after imbibition. The bars indicate the mean ±SD for each set of three independent experiments with significant difference at *P < 0.005 and **P < 0.001. (c) Root growth (left panel) and relative root growth (%, right panel) of plants used in (a) after treatment with various concentrations of NaCl. At least eight seedling roots were measured for each data point. Error bars indicate the mean ±SD for each set of three independent experiments with significant difference at *P < 0.005.

Figure 4

Overexpression of PI4Kγ3 enhances plant tolerance relatively to increased ABA concentrations. (a) Germination rates of the WT, pi4kγ3, 35S::PI4Kγ3/pi4kγ3 #1 and 35S::PI4Kγ3 #20 on MS medium with indicated ABA concentrations. Rates were scored from 1 day to 9 days after imbibition. The bars indicate the mean ±SD for each set of three independent experiments with significant difference at *P < 0.005 and **P < 0.001. (b) Root growth (left panel) and relative root growth (%, right panel) of plants used in (a) under indicated ABA concentration. Relative growth (%) was represented as the ratio to that of control. At least eight seedling roots were measured for each data point. Error bars indicate the mean ±SD for each set of three independent experiments with significant difference at *P < 0.005.

Figure 5

AtPI4Kγ3 positively regulates abiotic stress‐responsive genes. qRT‐PCR analysis of salt, ABA, cold and drought‐responsive marker genes using 2‐week‐old WT, pi4kγ3, 35S::PI4Kγ3/pi4kγ3 #1 and 35S::PI4Kγ3 #20 plants after indicated stress treatment. Error bars indicate the mean ±SD for each set of three independent experiments with significant difference at *P < 0.005 and **P < 0.001.

Figure 6

AtPI4Kγ3 is regulated by DNA methylation. (a) At5 g24240 harbours enriched methylating regions within its promoter. The figure shows a schematic representation of the gene structure (upper) and methylation status (lower) in wild‐type (WT) Col‐0 plants. (Upper) Green boxes, red boxes and thin lines between boxes depict exons, UTRs and introns, respectively. (Lower) Vertical lines reflect the methylation context as indicated colours. Data were obtained from the Arabidopsis epigenome map (Lister et al., 2008). Primers used for the chop–PCR are marked with arrows, R and F. (b) Chop–PCR analysis of gDNA from WT plants subjected to salt, ABA or without stresses (as control). Digestion with Hae III, MspI, Hpa II and McrBC was performed before PCR amplification. DNA without restriction enzymes (marked with ‐) was used as control.

Figure 7

The pi4kγ3 mutant shows increased sensitivity to H₂O₂. (a) Detached leaves from WT, pi4kγ3 and 35S::PI4Kγ3 #20 plants were incubated under white light for 3 days in 10 mm H₂O₂ solution, with or without pretreatment of 50 μm ABA for 3 h. (b) Resistance and chlorophyll content in plants used in (a) exposed to oxidative stress. Plants were grown on MS media for 5 days, and subsequently, transferred to new MS media containing 1 μm paraquat and grown for 1 week more (upper). Four‐day‐old seedlings of each transgenic plant were transferred to 3 μm paraquat solution for 3 days (lower). (c) Histochemical detection of H₂O₂ by 3, 3‐diaminobenzidine (DAB) in 7‐day‐old seedlings of plants used in (a) pretreated with 150 mm NaCl for 3 h.

Figure 8

AtPI4Kγ3 controls ROS accumulation. (a) Analysis of ROS production in roots during salt stress. Five‐day‐old seedlings of indicated Arabidopsis lines were exposed to 0.2 m NaCl for 10 min. Seedlings were stained with 2′, 7′‐dichlorodihydrofluorescein diacetate (H₂ DCFDA), and roots were observed by epifluorescence microscopy. Insets show corresponding bright field images. (b) Expression of RbohD, APX1, CAT1 and ZAT10 in the WT, pi4kγ3 and 35S::PI4Kγ3 #20 under salt stress. The bars indicate the mean ±SD for each set of three independent experiments with significant difference at *P < 0.005 and **P < 0.001.

Figure 9

AtPI4Kγ3 and AtbZIP60 are interdependent each other in the salt signalling pathway. Postgermination growth and germination rates (%) of WT, pi4kγ3, bzip60, pi4kγ3(±) bzip60 and pi4kγ3bzip60 (a), and WT, bzip60, 35S::PI4Kγ3 and bzip60 35S::PI4Kγ3 (b) on MS medium containing 125 mm NaCl. Photographs were taken at 7 days after imbibition. Germination rates (%) were calculated using the seeds, 1–5 days after imbibition. The bars indicate the mean ±SD for each set of three independent experiments with significant difference at *P < 0.005 and **P < 0.001. (c) Expression of salt‐responsive marker genes in WT, pi4kγ3, bzip60, pi4kγ3bzip60 and bzip60 35S::PI4Kγ3 by qRT‐PCR. Plants were subjected to liquid MS medium (control) or NaCl (150 mm). The bars indicate the mean ±SD for each set of three independent experiments with significant difference at *P < 0.005 and **P < 0.001.

Figure 10

UBL domains of AtPI4Kγ3 are important for salt stress resistance. (a) The schematic diagram represents the full‐length (F) as well as four truncated constructs of AtPI4Kγ3, such as deletion of UBL domains (ΔU), deletion of kinase domain (ΔK), deletion of kinase domain together with the middle region between kinase and 2nd UBL domain (ΔMK) and deletion of UBL domains together with the middle region (ΔUM) were used to fuse with vector for making transgenic plants. Corresponding total length (in aa., amino acid number) of the constructs are indicated at right side. (b) Germination rates and (c) postgermination growth of WT and transgenic plants containing constructs ΔUM, F, ΔK, ΔMK, ΔU and EV (empty vector) on MS medium containing 125 mm NaCl. Germination rates (%) were calculated using the seeds, 1–5 days after imbibition. Photograph was taken at 5 days after imbibition. The bars indicate the mean ±SD for each set of three independent experiments with significant difference at *P < 0.005 and **P < 0.001.

Figure 11

Overexpression of AtPI4Kγ3 delays flowering time under long‐day condition. (a) Flowering phenotype of WT, pi4kγ3 and 35S::PI4Kγ3 #20 plants grown under long‐day condition. Photograph was taken after 28 DAG. (b) Leaf number at flowering and time of bolting of the corresponding plants were measured. At least ten plants were selected to measure each data point. Error bars indicate the mean ±SD for each set of three independent experiments with significant difference at *P < 0.05 and **P < 0.01. (c) Expression analysis of the major flowering‐time regulators over 48 h under long‐day condition by qRT‐PCR in 2‐week‐old plants used in (a). The bars indicate the mean ±SD for each set of three independent experiments with significant difference at *P < 0.005 and **P < 0.001.

Figure 12

A proposed functional model for AtPI4Kγ3 in stress‐responsive and plant developmental pathways. The extracellular abiotic stresses induce ROS as a secondary signal molecule. AtPI4Kγ3 remains in inactive form due to hypermethylation under normal condition. Extracellular stress signal is sensed by hypermethylated AtPI4Kγ3 DNA in the nucleus (N) which becomes hypomethylated and starts transcription. Expression of AtPI4Kγ3 then activates ROS scavenging enzymes and suppresses ROS producer simultaneously. Thus, decreased toxic ROS accumulation finally may endow salt and ABA tolerance to plant. Under nonstressed condition, AtPI4Kγ3 activates some important negative regulators and suppresses some important positive regulators of flower transition, which may cause late flowering in Arabidopsis. The dashed arrows indicate possible regulation to control the ROS signalling pathways. PM, plasma membrane.