Wheat transcriptome profiling reveals abscisic and gibberellic acid treatments regulate early-stage phytohormone defense signaling, cell wall fortification, and metabolic switches following Fusarium graminearum-challenge.

BACKGROUND: Treatment of wheat with the phytohormones abscisic acid (ABA) and gibberellic acid (GA) has been shown to affect Fusarium head blight (FHB) disease severity. However, the molecular mechanisms underlying the elicited phenotypes remain unclear. Toward addressing this gap in our knowledge, global transcriptomic profiling was applied to the FHB-susceptible wheat cultivar 'Fielder' to map the regulatory responses effected upon treatment with ABA, an ABA receptor antagonist (AS6), or GA in the presence or absence of Fusarium graminearum (Fg) challenge. RESULTS: Spike treatments resulted in a total of 30,876 differentially expressed genes (DEGs) identified in 'Fielder' (26,004) and the Fg (4872) pathogen. Topology overlap and correlation analyses defined 9689 wheat DEGs as Fg-related across the treatments. Further enrichment analyses demonstrated that these included expression changes within 'Fielder' defense responses, cell structural metabolism, molecular transport, and membrane/lipid metabolism. Dysregulation of ABA and GA crosstalk arising from repression of 'Fielder' FUS3 was noted. As well, expression of a putative Fg ABA-biosynthetic cytochrome P450 was detected. The co-applied condition of Fg + ABA elicited further up-regulation of phytohormone biosynthesis, as well as SA and ET signaling pathways and cell wall/polyphenolic metabolism. In contrast, co-applied Fg + GA mainly suppressed phytohormone biosynthesis and signaling, while modulating primary and secondary metabolism and flowering. Unexpectedly, co-applied Fg + AS6 did not affect ABA biosynthesis or signaling, but rather elicited antagonistic responses tied to stress, phytohormone transport, and FHB disease-related genes. CONCLUSIONS: Observed exacerbation (misregulation) of classical defense mechanisms and cell wall fortifications upon ABA treatment are consistent with its ability to promote FHB severity and its proposed role as a fungal effector. In contrast, GA was found to modulate primary and secondary metabolism, suggesting a general metabolic shift underlying its reduction in FHB severity. While AS6 did not antagonize traditional ABA pathways, its impact on host defense and Fg responses imply potential for future investigation. Overall, by comparing these findings to those previously reported for four additional plant genotypes, an additive model of the wheat-Fg interaction is proposed in the context of phytohormone responses.

The malate-activated ALMT12 anion channel in the grass Brachypodium distachyon is co-activated by Ca2+/calmodulin.

In plants, strict regulation of stomatal pores is critical for modulation of CO2 fixation and transpiration. Under certain abiotic and biotic stressors, pore closure is initiated through anionic flux, with calcium (Ca2+) playing a central role. The aluminum-activated malate transporter 12 (ALMT12) is a malate-activated, voltage-dependent member of the aluminum-activated malate transporter family that has been implicated in anionic flux from guard cells controlling the stomatal aperture. Herein, we report the characterization of the regulatory mechanisms mediating channel activities of an ALMT from the grass Brachypodium distachyon (BdALMT12) that has the highest sequence identity to Arabidopsis thaliana ALMT12. Electrophysiological studies in a heterologous cell system confirmed that this channel is malate- and voltage-dependent. However, this was shown to be true only in the presence of Ca2+ Although a general kinase inhibitor increased the current density of BdALMT12, a calmodulin (CaM) inhibitor reduced the Ca2+-dependent channel activation. We investigated the physiological relevance of the CaM-based regulation in planta, where stomatal closure, induced by exogenous Ca2+ ionophore and malate, was shown to be inhibited by exogenous application of a CaM inhibitor. Subsequent analyses revealed that the double substitutions R335A/R338A and R335A/K342A, within a predicted BdALMT12 CaM-binding domain (CBD), also decreased the channels' ability to activate. Using isothermal titration calorimetry and CBD-mimetic peptides, as well as CaM-agarose affinity pulldown of full-length recombinant BdALMT12, we confirmed the physical interaction between the CBD and CaM. Together, these findings support a co-regulatory mechanism of BdALMT12 activation by malate, and Ca2+/CaM, emphasizing that a complex regulatory network modulates BdALMT12 activity.

Identification and characterization of interactions between abscisic acid and human heat shock protein 70 family members.

Abscisic acid (ABA) is a stress-inducible plant hormone comprising an inevitable component of the human diet. Recently, stress-induced accumulation of autocrine ABA was shown in humans, as well as ABA-mediated modulation of a number of disease-associated systems. Now, the application of a chemical proteomics approach to gain further insight into ABA mechanisms of action in mammalian cells is reported. An ABA mimetic photoaffinity probe was applied to intact mammalian insulinoma and embryonic cells, leading to the identification of heat shock protein 70 (HSP70) family members, (including GRP78 and HSP70-2) as putative human ABA-binding proteins. In vitro characterization of the ABA-HSP70 interactions yielded K(d)s in the 20-60 µM range, which decreased several fold in the presence of co-chaperone. However, ABA was found to have only variable- and co-chaperone-independent effects on the ATPase activity of these proteins. The potential implications of these ABA-HSP70 interactions are discussed with respect to the intracellular protein folding and extracellular receptor-like activities of these stress-inducible proteins. While mechanistic and functional relevance remain enigmatic, we conclude that ABA can bind to human HSP70 family members with physiologically relevant affinities and in a co-chaperone-dependent manner.

Identification and characterization of interactions between abscisic acid and mitochondrial adenine nucleotide translocators.

ABA (abscisic acid) is a plant hormone involved in important processes including development and stress responses. Recent reports have identified a number of plant ABA receptors and transporters, highlighting novel mechanisms of ABA action. In the present paper we describe application of a chemical proteomics approach leading to the identification of mitochondrial ANTs (adenine nucleotide translocators) as ABA-interacting proteins. Initial in vitro studies confirmed inhibition of ANT-dependent ATP translocation by ABA. Further analysis demonstrated ANT-dependent uptake of ABA into both recombinant Arabidopsis thaliana ANT2-containing proteoliposomes and native isolated spinach mitochondria; the latter with a Km of 3.5 µM and a Vmax of 2.5 nmol/min per g of protein. ATP was found to inhibit ANT-dependent ABA translocation. Specificity profiles highlight the possibility of mechanistic differences in translocation of ABA and ATP. Finally, ABA was shown to stimulate ATPase activity in spinach mitochondrial extracts. ABA concentrations in plant cells are estimated to reach the low micromolar range during stress responses, supporting potential physiological relevance of these in vitro findings. Overall, the present in vitro work suggests the possibility of as yet uncharacterized mechanisms of ABA action in planta related to inhibition of mitochondrial ATP translocation and functional localization of ABA in the mitochondrial matrix.

In vitro characterization of ligand-induced oligomerization of the S. cerevisiae G-protein coupled receptor, Ste2p.

BACKGROUND: The S. cerevisiae alpha-factor receptor, Ste2p, is a G-protein coupled receptor that plays key roles in yeast signaling and mating. Oligomerization of Ste2p has previously been shown to be important for intracellular trafficking, receptor processing and endocytosis. However the role of ligand in receptor oligomerization remains enigmatic. METHODS: Using functional recombinant forms of purified Ste2p, atomic force microscopy, dynamic light scattering and chemical crosslinking are applied to investigate the role of ligand in Ste2p oligomerization. RESULTS: Atomic force microscopy images indicate a molecular height for recombinant Ste2p in the presence of alpha-factor nearly double that of Ste2p alone. This observation is supported by complementary dynamic light scattering measurements which indicate a ligand-induced increase in the polydispersity of the Ste2p hydrodynamic radius. Finally, chemical cross-linking of HEK293 plasma membranes presenting recombinant Ste2p indicates alpha-factor induced stabilization of the dimeric form and higher order oligomeric forms of the receptor upon SDS-PAGE analysis. CONCLUSIONS: alpha-factor induces oligomerization of Ste2p in vitro and in membrane. GENERAL SIGNIFICANCE: These results provide additional evidence of a possible role for ligand in mediation of Ste2p oligomerization in vivo.

Identification and characterization of a unique cysteine residue proximal to the catalytic site of Arabidopsis thaliana carotenoid cleavage enzyme 1.

AtCCD1 and AtNCED3 are related carotenoid cleavage enzymes from Arabidopsis thaliana that catalyze the oxidative cleavage of, respectively, the 9,10 (9',10') double bonds of carotenoid substrates such as beta-carotene, and the 11,12 double bond of 9-cis epoxycarotenoids. Although the cellular and cleavage functionalities of these enzymes have been reported, their mechanisms and related structural environments mediating these disparate specificities in homologous enzymes have not been well characterized. By relating the differences observed in UV and visible light absorption and Cu(II) electron paramagnetic signals to variations in sequence alignments and 3-D homology models of the two A. thaliana enzymes, we identified a putatively proximal cysteine residue (Cys352) in AtCCD1 that is not conserved in AtNCED3. Spectral analysis of the Cys to Ala mutant confirmed its uniqueness and proximity to the metal binding site, but precluded any role for the residue in the mediation of the observed metal binding affinity or associated steric constraint differences. Further analysis of kinetic substrate cleavage properties indicated a decrease in Vmax and a subtle increase in Km for the C352A mutant compared with those observed for the wild-type, thus confirming catalytic site proximity and suggesting possible roles for the unique cysteine in the modulation of substrate affinity and (or) the reaction rate of AtCCD1.

Purification and characterization of a recombinant G-protein-coupled receptor, Saccharomyces cerevisiae Ste2p, transiently expressed in HEK293 EBNA1 cells.

The production of milligram quantities of purified, active, folded membrane protein from heterologous expression systems remains a general challenge due to intrinsically low expression levels, misfolding, and instability. Here we report the overexpression and purification of milligram quantities of functional Saccharomyces cerevisiae G-protein-coupled receptor, Ste2p, from transiently transfected human embryonic kidney 293 EBNA1 cells. Fluorescent microscopy indicates localization of Ste2p-GFP and Fc-Ste2p-GFP fusion receptors to the cell membrane. Up to 2 mg (approximately 10 pmol/million cells) of the Fc-Ste2p-GFP fusion and 1 mg of a Ste2p-Strep-TagII/(His)8-tagged version were purified per liter of culture following protein A-Sepharose and Talon metal affinity chromatography, respectively. Two distinct fluorescent labels, the hydrophobic 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) and the more hydrophilic fluorescein-5-maleimide (FM), were individually attached to the C-terminus of the alpha-mating factor ligand by addition of a reactive cysteine residue to produce active fluorescent pheromones. In vitro fluorescent ligand binding assays demonstrated that a high percentage of the recombinant purified receptor is correctly folded and able to bind ligand. KD values of 34 +/- 3 and 300 +/- 20 nM were observed respectively for the CPM- and FM-labeled ligands. These results combined with blue-shifted emission peaks and loss of fluorescent quenching observed for both fluorescent-labeled Cys alpha-factors when bound to receptor support a model in which the C-terminus of the ligand is packed in a hydrophobic pocket at the interface between the transmembrane and extracellular loop domains. Overall, we present an efficient system for recombinant production of milligram quantities of purified Ste2p in a biologically active form with applications to future structure and functional studies.

NMR spectroscopy of phosphorylated wild-type rhodopsin: mobility of the phosphorylated C-terminus of rhodopsin in the dark and upon light activation.

Binding of arrestin to light-activated rhodopsin involves recognition of the phosphorylated C-terminus and several residues on the cytoplasmic surface of the receptor. These sites are in close proximity in dark, unphosphorylated rhodopsin. To address the position and mobility of the phosphorylated C-terminus in the active and inactive receptor, we combined high-resolution solution and solid state NMR spectroscopy of the intact mammalian photoreceptor rhodopsin in detergent micelles as a function of temperature. The (31)P NMR resonance of rhodopsin phosphorylated by rhodopsin kinase at the C-terminal tail was observable with single pulse excitation using magic angle spinning until the sample temperature reached -40 degrees C. Below this temperature, the (31)P resonance broadened and was only observable using cross polarization. These results indicate that the phosphorylated C-terminus is highly mobile above -40 degrees C and immobilized at lower temperature. To probe the relative position of the immobilized phosphorylated C-terminus with respect to the cytoplasmic domain of rhodopsin, (19)F labels were introduced at positions 140 and 316 by the reaction of rhodopsin with 2,2,2-trifluoroethanethiol (TET). Solid state rotational-echo double-resonance (REDOR) NMR was used to probe the internuclear distance between the (19)F and the (31)P-labels. The REDOR technique allows (19)F...(31)P distances to be measured out to approximately 12 A with high resolution, but no significant dephasing was observed in the REDOR experiment in the dark or upon light activation. This result indicates that the distances between the phosphorylated sites on the C-terminus and the (19)F sites on helix 8 (Cys 316) and in the second cytoplasmic loop (Cys140) are greater than 12 A in phosphorylated rhodopsin.