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. 2015 Apr;27(4):1082-97.
doi: 10.1105/tpc.114.135160. Epub 2015 Apr 21.

VIH2 Regulates the Synthesis of Inositol Pyrophosphate InsP8 and Jasmonate-Dependent Defenses in Arabidopsis

Debabrata Laha  1 , Philipp Johnen  1 , Cristina Azevedo  2 , Marek Dynowski  3 , Michael Weiß  4 , Samanta Capolicchio  5 , Haibin Mao  6 , Tim Iven  7 , Merel Steenbergen  8 , Marc Freyer  1 , Philipp Gaugler  1 , Marília K F de Campos  1 , Ning Zheng  6 , Ivo Feussner  7 , Henning J Jessen  5 , Saskia C M Van Wees  8 , Adolfo Saiardi  2 , Gabriel Schaaf  9
Affiliations

VIH2 Regulates the Synthesis of Inositol Pyrophosphate InsP8 and Jasmonate-Dependent Defenses in Arabidopsis

Debabrata Laha et al. Plant Cell. 2015 Apr.

Abstract

Diphosphorylated inositol polyphosphates, also referred to as inositol pyrophosphates, are important signaling molecules that regulate critical cellular activities in many eukaryotic organisms, such as membrane trafficking, telomere maintenance, ribosome biogenesis, and apoptosis. In mammals and fungi, two distinct classes of inositol phosphate kinases mediate biosynthesis of inositol pyrophosphates: Kcs1/IP6K- and Vip1/PPIP5K-like proteins. Here, we report that PPIP5K homologs are widely distributed in plants and that Arabidopsis thaliana VIH1 and VIH2 are functional PPIP5K enzymes. We show a specific induction of inositol pyrophosphate InsP8 by jasmonate and demonstrate that steady state and jasmonate-induced pools of InsP8 in Arabidopsis seedlings depend on VIH2. We identify a role of VIH2 in regulating jasmonate perception and plant defenses against herbivorous insects and necrotrophic fungi. In silico docking experiments and radioligand binding-based reconstitution assays show high-affinity binding of inositol pyrophosphates to the F-box protein COI1-JAZ jasmonate coreceptor complex and suggest that coincidence detection of jasmonate and InsP8 by COI1-JAZ is a critical component in jasmonate-regulated defenses.

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Figures

Figure 1.
Figure 1.
Vip1/PPIP5K Homologs Are Ubiquitous in Plants. BLAST search analyses were performed employing the protein sequence of the Saccharomyces cerevisiae Vip1 ATP-grasp kinase domain (residues 1 to 535) as a search template. The tree was estimated from an alignment of ATP-grasp kinase domains of the encoded proteins using maximum likelihood. Branch support was calculated from 10,000 bootstrap replicates, and values below 50% are omitted. Branch lengths are given in terms of expected numbers of amino acid substitutions per site.
Figure 2.
Figure 2.
VIH1 and VIH2 Are Functional Vip1-Type PPIP5 Kinases. (A) and (B) Structural model of the VIH2 ATP-grasp kinase domain (left) and the hPPIP5K2 ATP-grasp kinase domain (PDB ID: 3T9D, right) depicting the 5-InsP7 binding sites and key catalytic residues. Residues coordinating substrate via polar contacts are shown as sticks, polar interactions are highlighted by dashed lines, α-helices are rendered in blue, β-sheets in orange, substrate (5-InsP7) is rendered in magenta, and Mg2+ ions are presented as green spheres. Three carbon atoms on the inositol ring are numbered. The ATP analog AMP-PNP (in [B]) is depicted with gray carbon and orange and red phosphate groups. (C) and (D) Complementation of vip1Δ-associated growth defects in yeast by ectopic expression of inositol pyrophosphate synthetases. The vip1Δ yeast strain transformed with episomal pDR195(URA3) plasmids carrying either VIP1, VIH1, or VIH2, sequences encoding their respective ATP-grasp kinase domains (KD) or designated kinase domain mutants, or carrying KCS1 were spotted in 8-fold serial dilutions onto uracil-free minimal medium in presence or absence of 6-azauracil, as indicated. Rescue on medium supplemented with 6-azauracil (right) reports Vip1 activity. (E) and (F) Normalized HPLC profiles of inositol phosphates of extracts from designated [3H] inositol-labeled yeast transformants. Extracts were resolved by Partisphere SAX HPLC and fractions collected each minute for subsequent determination of radioactivity as indicated. Changes in elution times in independent experiments were observed and can be explained by subtle changes in column properties or column change. Experiments were repeated three times with similar results. (G) Complementation assays of kcs1Δ-associated growth defects on high salt by ectopic expression inositol pyrophosphate synthetases. Wild-type (wt) or kcs1Δ yeast transformants (both DDY1810 background) carrying designated plasmids were spotted in 8-fold serial dilutions onto solid minimal media (MM, uracil deficient CSM media with YNB and appropriate supplements) in presence or absence of NaCl and onto solid YPDA media incubated at 37°C.
Figure 3.
Figure 3.
Expression Analyses Suggest Specialized Functions of VIH1 and VIH2, and Inositol Pyrophosphates Can Be Detected in Arabidopsis Extracts and Are Regulated by Jasmonate. (A) and (B) qPCR analyses of VIH expression in Col-0 plants using cDNA prepared from RNA extracts of different plant tissues as indicated. Averages of triplicate reactions ± sd are shown. β-TUBULIN was used as reference gene. Transcript levels of VIH1 and VIH2 are presented relative to β-TUBULIN transcript. The experiment was repeated three times with similar results. (C) and (D) MeJA increases InsP8 level. Normalized HPLC profiles (C) of 3-week old [3H] inositol-labeled Col-0 seedlings that were untreated (solid gray line) or treated for 4 h with 50 μM MeJA (solid red lines). Treated and nontreated plants were harvested simultaneously to avoid daytime-dependent differences in inositol polyphosphate homeostasis. The experiment was repeated with similar results, and representative results from one experiment are shown. For relative amounts of respective species (D), averages of fold differences after MeJA treatment of three independent experiments ± se are shown. Asterisks indicate statistical differences (Student’s t test; *P < 0.02).The isomeric identity of InsP5b is unknown in Arabidopsis seedlings. Based on chromatographic mobilities presented in a previous study on seedlings of Col-0 plants and ipk1-1 plants (Stevenson-Paulik et al., 2005), and comparison with chromatographic mobilities of inositol polyphosphates in the same ipk1-1 line on our HPLC (Supplemental Figures 8A and 8B), InsP5a represents Ins(1,3,4,5,6)P5 and InsP5c represents Ins(1,2,4,5,6)P5 or its enatiomer Ins(2,3,4,5,6)P5.
Figure 4.
Figure 4.
Bulk Steady State and Jasmonate-Induced Pools of InsP8 in Arabidopsis Seedlings Depend on VIH2. Normalized HPLC profiles ([A] to [C]) or relative amounts (D) of inositol phosphate species of 3-week-old [3H] inositol-labeled Col-0 (solid black line) and vih2-4 seedlings. In (B) and (C), plants were treated with 50 µM MeJA and harvested after 30 min together with nontreated plants. Extracts were resolved by Partisphere SAX HPLC and fractions collected each minute for subsequent determination of radioactivity. The experiment was repeated with similar results, and representative results from one experiment are shown. (B) is a zoom-in into the InsP5 (left) and InsP6-8 (right) regions of HPLC runs with extracts of MeJA-treated Col-0 and vih2 seedlings as indicated. For InsP5a-c isomer identities, see comment in Figure 3. (C) is a zoom-in into the InsP6-8 regions of HPLC runs with extracts of Col-0 (left) and vih2 (right) seedlings with or without MeJA treatment as indicated. For relative amounts (D), data are presented either as InsP7/InsP6 ratio (a measure of IP6K activity) or as InsP8/InsP7 ratio (a measure of PPIP5K activity). The data represent means ± se.
Figure 5.
Figure 5.
Arabidopsis vih2 Lines Have Reduced Defenses against Larvae of Herbivorous Insects and Are Compromised in Jasmonate Perception. (A) and (B) Larval development was monitored in a no choice setup. One caterpillar each (larval stage L1) of the Brassicaceae specialist P. rapae (A) or the generalist M. brassicae (B) was released onto a single 5-week-old plant (n = 20) of the designated genotype. Fresh weight of caterpillars was determined after 7 d (P. rapae) or 8 d (M. brassicae). The values represent means ± se. Asterisks indicate statistical differences (Student’s t test; *P < 0.02). Plant genotype-dependent size differences of M. brassicae larvae are also visualized by a photograph ([B], right panel). Experiments were repeated with similar results. (C) and (D) Determination of bioactive conjugates JA-Val and JA-Ile/Leu. Conjugate levels were determined in rosette leaves of 4-week-old plants of designated genotypes under sterile conditions and 3 h after inflecting wounding by squeezing each leaf with forceps. Data represent means of three independent biological replica ± sd. Statistical significance is indicated by asterisks (Student’s t test; *P < 0.02 and **P < 0.005). (E) qPCR analysis of JA-dependent genes. Gene expression was analyzed by qPCR analyses using RNA extracted from pooled leaves (n = 5) of 5-week-old plants of the designated genotype that were untreated or infested for 24 h by P. rapae larvae as indicated. PP2AA3 was used as a reference gene. The expression value of untreated Col-0 was set to 1. Shown are means ± se (n = 3). qPCR analyses were repeated with similar results.
Figure 6.
Figure 6.
Structural Models of ASK1-COI1-JAZ1-Coronatine in Complex with Ins(1,2,4,5,6)P5 or 1,5-InsP8 and Functional Evaluation of Proposed 1,5-InsP8 Binding Mutants Suggest a Role of InsP8 in Jasmonate Receptor Complex Formation. (A) and (B) COI1-JAZ structures containing Ins(1,2,4,5,6)P5 or 1,5-InsP8 as obtained from in silico docking experiments are shown. COI1 (gray ribbon), coronatine (COR) in yellow stick representation, and inositol polyphosphates (rendered as stick in magenta) are presented. Hydrogen bonds and salt bridge networks are depicted as dashed lines. Residues in bold were substituted by Ile for yeast two-hybrid studies. (C) JAZ1 interaction with wild-type or mutant COI1 in yeast was evaluated in the presence of 50 μM coronatine by coexpression of pGBKT7-COI1 (and mutated versions as indicated) with pGADT7-JAZ1 in yeast strain Y187 (Clontech) and subsequent quantification of β-galactosidase-mediated hydrolysis of ortho-nitrophenyl-β-d-galactopyranoside. Values represent means of four independent biological replica ± sd. (D) Stability of mutant COI1 protein. Immunoblots of soluble lysates prepared from tobacco (Nicotiana benthamiana) leaves expressing COI1 mutants (as designated) in translational fusion with N-terminal GST. Equal amounts of total protein were loaded, and COI1 was detected with antibodies against GST (Sigma-Aldrich). As a normalization control (lower panel), a representative unspecific band was chosen.
Figure 7.
Figure 7.
Plant Inositol Pyrophosphates Are Superior Ligands of the ASK1-COI1-JAZ1 Complex Compared with Less Anionic Inositol Polyphosphate Species. Direct binding of [3H]-InsP5, [3H]-InsP6, and [3H]-InsP7 (purified and desalted from [3H]-myo-inositol labeled seedlings of the ipk1-1 mutant [InsP5] or Col-0 seedlings [InsP6 and InsP7]) to the ASK1/COI1/His8-MBP-JAZ1 jasmonate receptor complex or to individual components of the receptor complex (ASK1-COI1 or His8-MBP-JAZ1) was analyzed with or without 1 μM coronatine. A total activity of 2000 dpm was used for each [3H]-labeled inositol phosphate species. The average of recovered radiolabel with [3H]-InsP7 in (B) is set to 100%. Values show means ± se (n = 2 or 3) of radiolabel recovered by pull-down of His8-MBP-JAZ1 via metal affinity chromatography, and experiments were repeated with similar results.
Figure 8.
Figure 8.
Model of the Role of VIH2 and InsP8 in the Wound Response. Mechanical wounding or herbivore attack stimulate the synthesis of JA and bioactive JA conjugates such as JA-Ile. Increasing jasmonate levels trigger a fast VIH2-dependent increase in InsP8, which is most likely caused by posttranslational activation of the VIH2 protein. Both JA-Ile and InsP8 occupy designated binding pockets in COI1-ASK1 and might work as molecular glue to recruit the JAZ repressor protein. Subsequent polyubiquitylation of JAZ by the SCF ubiquitin E3 ligase complex causes proteasomal degradation of the JAZ repressor and allows expression of jasmonate/InsP8-responsive genes such as VSP2. The physiological role of other inositol polyphosphates on potentiating jasmonate dependent formation of the SCFCOI1 ubiquitin E3 ligase complex remains unclear.
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