Inhibition of cryoaggregation of phospholipid liposomes by an Arabidopsis intrinsically disordered dehydrin and its K-segment.

Dehydrin is an intrinsically disordered protein involved in the cold tolerance of plants. Although dehydrins have been thought to protect biomembranes under cold conditions, the underlying protective mechanism has not been confirmed. Here we report that Arabidopsis dehydrin AtHIRD11 inhibited the aggregation of phospholipid liposomes after freezing and thawing. AtHIRD11 showed significantly greater cryoaggregation-prevention activity than cryoprotective agents such as trehalose, proline, and polyethylene glycols. Amino acid sequence segmentation analysis indicated that the K-segment of AtHIRD11 inhibited the cryoaggregation of phosphatidylcholine (PC) liposomes but other segments did not. This showed that K-segments conserved in all dehydrins were likely to be the cryoprotective sites of dehydrins. Amino acid replacement for a typical K-segment (TypK for short) sequence demonstrated that both hydrophobic and charged amino acids were required for the cryoaggregation-prevention activity of PC liposomes. The amino acid shuffling of TypK remarkably reduced cryoprotective activity. Although TypK did not bind to PC liposomes in solution, the addition of liposomes reduced its disordered content under crowded conditions. Together, these results suggested that dehydrins protected biomembranes via conserved K-segments whose sequences were optimized for cryoprotective activities.

An intrinsically disordered radish vacuolar calcium-binding protein (RVCaB) showed cryoprotective activity for lactate dehydrogenase with its hydrophobic region.

A soluble protein fraction from radish (Raphanus sativus L.) taproot had cryoprotective activity for lactate dehydrogenase (LDH). The activity was found mainly in the heat-stable fractions of soluble proteins. The cryoprotective protein, whose molecular mass was 43 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis, was obtained by successive chromatographies on TOYOPEARL SuperQ and TOYOPEARL DEAE. MALDI-TOF MS/MS analysis indicated that the purified protein was a radish vacuolar calcium-binding protein (RVCaB), which is reportedly related to calcium storage in the vacuoles of radish taproot. The purified RVCaB inhibited the cryoinactivation, cryodenaturation, and cryoaggregation of LDH. RVCaB had greater cryoprotective activity than general cryoprotectants. When RVCaB was divided into 15 segments (Seg01 to Seg15, 15 amino acids each), Seg03, which had a high hydrophobicity scale, showed remarkable cryoprotective activity. This indicated that RVCaB protected LDH from freezing and thawing damage presumably through a specific hydrophobic area (i.e., Seg03).

Potential use of essential oils to enhance heat tolerance in plants.

Isothiocyanates, monoterpenes, and leaf volatiles that are components of essential oils induce the expression of heat shock protein genes in plant systems. Here, the modes of heat shock responses induced by the essential oil compounds and their heat-tolerance-enhancing activities are described. Traditionally, green manure produced from essential-oil-containing plants has been used because such manure is thought to have beneficial effects in fertilizing, allelopathic, antibacterial, and animal-repellent activities. In addition to these effects, stress (especially heat stress)-tolerance-enhancing activities can be expected. Biostimulants containing such essential oils may be able to maintain the yield and quality of crops under increasing ambient temperatures. In this review, chemicals that enhance the heat tolerance of plants are designated as heat tolerance enhancers (HTLEs). Some essential oil compounds can be categorized as HTLEs available for biostimulants.

Cryoprotective activity of Arabidopsis KS-type dehydrin depends on the hydrophobic amino acids of two active segments.

Dehydrins are intrinsically disordered proteins which are related to cold tolerance in plants. Dehydrins show potent cryoprotective activities for freeze-sensitive enzymes such as lactate dehydrogenase (LDH). Previous studies demonstrated that K-segments conserved in dehydrins had cryoprotective activities and that K-segment activities depended on the hydrophobic amino acids in the segment. However, the cryoprotective roles of hydrophobic amino acids in dehydrin itself have not been reported. Here, we demonstrated that hydrophobic amino acids were required for the cryoprotective activity of Arabidopsis dehydrin AtHIRD11. Cryoprotective activities were compared between AtHIRD11 and the corresponding mutant in which all hydrophobic residues were changed to T (AtHIRD11Φ/T) by using LDH. The change strikingly reduced AtHIRD11 activity. A segmentation analysis indicated that the conserved K-segment (Kseg) and a previously unidentified segment (non-K-segment 1, NK1) showed cryoprotective activities. Circular dichroism indicated that the secondary structures of all peptides showed disorder, but only cryoprotective peptides changed to the ordered forms by sodium dodecyl sulfate. Ultracentrifuge analysis indicated that AtHIRD11 and AtHIRD11Φ/T had similar molecular sizes in solution. These results suggest that not only structural disorder but also hydrophobic amino acids contributed to the cryoprotective activity of AtHIRD11. A possible mechanism based on an extended molecular shield model is proposed.

F-segments of Arabidopsis dehydrins show cryoprotective activities for lactate dehydrogenase depending on the hydrophobic residues.

Although dehydrins show cryoprotective activities for freeze-sensitive enzymes, the underlying mechanism is still under investigation. Here, we report that F-segments conserved in some dehydrins cryoprotected lactate dehydrogenase (LDH) as well as K-segments, which were previously identified as cryoprotective segments of dehydrins. The cryoprotective activity levels of four F-segments of Arabidopsis dehydrins were similar to that of a typical K-segment. Amino acid substitution experiments indicated that the activity of the F-segment of Arabidopsis COR47 (designated as Fseg) depended on the hydrophobic residues (L, F, and V). Intriguingly, when all the amino acids other than the hydrophobic residues were changed to glycine, the cryoprotective activity did not change, suggesting that the hydrophobic amino acids were sufficient for Fseg activity. Circular dichroism analysis indicated that Fseg was mainly disordered in aqueous solution as well as Fseg_Φ/T, in which the hydrophobic residues of Fseg were changed to T. This suggested that the hydrophobic interaction might be related to the cryoprotective activities of Fseg.

Induction of the heat shock response in Arabidopsis by heat shock protein 70 inhibitor VER-155008.

The heat shock protein 90 (HSP90) inhibitor, geldanamycin, is a chemical inducer of the heat shock response (HSR) in Arabidopsis. Geldanamycin is thought to activate the heat shock signal by dissociating the HSP90-heat shock factor (HSF) complex. Recent studies have indicated that plant HSP70 is also associated with HSF, suggesting that inhibition of HSP70 may induce the HSR. However, no studies have been conducted to test this hypothesis. Here, we found that a specific HSP70 inhibitor VER-155008 activated the promoter of a small HSP gene (At1 g53540, HSP17.6C-CI) of Arabidopsis, which was shown to be activated by geldanamycin and other HSP90 inhibitors. The production of HSP17.6C-CI, HSP70 and HSP90.1 proteins in Arabidopsis was enhanced by the addition of VER-155008. The reduction of chlorophyll contents by heat shock was ameliorated by VER-155008. Chaperone analyses indicated that VER-155008 inhibited the chaperone activities of wheat germ extract and human HSP70/HSP40, respectively. These results suggest that the inhibition of HSP70 by VER-155008 enhanced the heat tolerance of Arabidopsis by inducing the HSR in the plant.

Monoterpenes induce the heat shock response in Arabidopsis.

Monoterpenes are common constituents of essential oils produced by plants. Although it has been reported that monoterpenes enhanced the heat tolerance of plants, the mechanism has not been elucidated. Here, we tested whether 13 monoterpenes promoted the heat shock response (HSR) in Arabidopsis. To assess the HSR-inducing activity of monoterpenes, we produced transgenic Arabidopsis, which has the ß-glucuronidase gene driven by the promoter of a small heat shock protein (HSP17.6C-CI) gene. Results indicated that two monocyclic and four bicyclic monoterpenes showed HSR-inducing activities using the reporter gene system. In particular, (-)-perillaldehyde, which is a monocyclic monoterpene, demonstrated the most potent HSR-inducing activity. (-)-Perillaldehyde significantly inhibited the reduction of chlorophyll content by heat shock in Arabidopsis seedlings. Our previous study indicated that chemical HSR inducers such as geldanamycin and sanguinarine inhibited the activity of plant chaperones in vitro. (-)-Perillaldehyde also inhibited chaperone activity, indicating that it might promote the expression of heat shock protein genes by inhibiting chaperones in the plant cell.

Metal ion - Dehydrin interactions investigated by affinity capillary electrophoresis and computer models.

Dehydrins are specialized proteins which are related to environmental stress tolerance in plants. The proteins can bind different metal ions and have versatile other functions such as reduction of reactive oxygen species and acting as transcription factor. The structure determination of proteins from this family is challenging, since they have a high number of disordered structure elements. Consequently, to determine the functionality of these proteins on a molecular basis a computed model is helpful. This work focuses on a model for the Arabidopsis thaliana dehydrin AtHIRD11. To develop a model which reflects experimental data from literature and own binding data from affinity capillary electrophoresis experiments, a more rigid state of this protein was chosen. The Cu2+-complex of this protein was formed and evaluated. The model explains some of the properties of the complexes. Possible Cu2+-bindings site were found and the change of conformations were investigated via molecular dynamics simulation. The AtHIRD11-Cu2+-complex is a first approach towards a complex model for a structural versatile protein, which is already sufficient to explain binding data and possible structure elements.

Hyperthermia and chemotherapy using Fe(Salen) nanoparticles might impact glioblastoma treatment.

We previously reported that µ-oxo N,N'-bis(salicylidene)ethylenediamine iron [Fe(Salen)], a magnetic organic compound, has direct anti-tumor activity, and generates heat in an alternating magnetic field (AMF). We showed that Fe(Salen) nanoparticles are useful for combined hyperthermia-chemotherapy of tongue cancer. Here, we have examined the effect of Fe(Salen) on human glioblastoma (GB). Fe(Salen) showed in vitro anti-tumor activity towards several human GB cell lines. It inhibited cell proliferation, and its apoptosis-inducing activity was greater than that of clinically used drugs. Fe(Salen) also showed in vivo anti-tumor activity in the mouse brain. We evaluated the drug distribution and systemic side effects of intracerebrally injected Fe(Salen) nanoparticles in rats. Further, to examine whether hyperthermia, which was induced by exposing Fe(Salen) nanoparticles to AMF, enhanced the intrinsic anti-tumor effect of Fe(Salen), we used a mouse model grafted with U251 cells on the left leg. Fe(Salen), BCNU, or normal saline was injected into the tumor in the presence or absence of AMF exposure. The combination of Fe(Salen) injection and AMF exposure showed a greater anti-tumor effect than did either Fe(Salen) or BCNU alone. Our results indicate that hyperthermia and chemotherapy with single-drug nanoparticles could be done for GB treatment.

The role of hydrophobic amino acids of K-segments in the cryoprotection of lactate dehydrogenase by dehydrins.

Dehydrins, which are group 2 late embryogenesis abundant (LEA) proteins, accumulate in plants during the development of the embryo and exposure to abiotic stresses including low temperature. Dehydrins exhibit cryoprotection of freezing-sensitive enzymes, e.g. lactate dehydrogenase (LDH). Although it has been reported that K-segments conserved in dehydrins are related to their cryoprotection activity, it has not been determined which sequence features of the K-segments contribute to the cryoprotection. A cryoprotection assay using LDH indicated that 13 K-segments including 12 K-segments found in Arabidopsis dehydrins and a typical K-segment (TypK, EKKGIMEKIKEKLPG) derived from the K-segments of many plants showed similar cryoprotective activities. Mutation of the TypK sequence demonstrated that hydrophobic amino acids were clearly involved in preventing the cryoinactivation, cryoaggregation, and cryodenaturation of LDH. We propose that the cryoprotective activities of dehydrins may be made possible by the hydrophobic residues of the K-segments.

The Arabidopsis KS-type dehydrin recovers lactate dehydrogenase activity inhibited by copper with the contribution of His residues.

Dehydrin, which is one of the late embryogenesis abundant (LEA) proteins, is involved in the ability of plants to tolerate the lack of water. Although many reports have indicated that dehydrins bind heavy metals, the physiological role of this metal binding has not been well understood. Here, we report that the Arabidopsis KS-type dehydrin (AtHIRD11) recovered the lactate dehydrogenase (LDH) activity denatured by Cu(2+). The LDH activity was partially inhibited by 0.93 µM Cu(2+) but totally inactivated by 9.3 µM Cu(2+). AtHIRD11 recovered the activity of LDH treated with 9.3 µM Cu(2+) in a dose-dependent manner. The recovery activity of AtHIRD11 was significantly higher than those of serum albumin and lysozyme. The conversion of His residues to Ala in AtHIRD11 resulted in the loss of the Cu(2+) binding of the protein as well as the disappearance of the conformational change induced by Cu(2+) that is observed by circular dichroism spectroscopy. The mutant protein showed lower recovery activity than the original AtHIRD11. These results indicate that AtHIRD11 can reactivate LDH inhibited by Cu(2+) via the His residues. This function may prevent physiological damage to plants due to heavy-metal stress.

The isoquinoline alkaloid sanguinarine which inhibits chaperone activity enhances the production of heat shock proteins in Arabidopsis.

Sanguinarine is an isoquinoline alkaloid produced by Papaveraceae plants. Because sanguinarine has antimicrobial activity, it is believed to be related to the plants' chemical defense systems. However, its action against plants has not been well understood. A previous study reported that among 12 alkaloids, sanguinarine was the only compound which enhanced heat tolerance in Arabidopsis. Here we performed a promoter assay using a heat shock protein gene (HSP17.6C-CI) of Arabidopsis to assess the induction of heat shock responses by alkaloids. Although sanguinarine induced the heat shock response, the other 11 alkaloids did not. Sanguinarine promoted the production of HSP17.6C-CI protein, but berberine and papaverine, which are isoquinoline alkaloids as well as sanguinarine, did not promote it. It is known that geldanamycin, a small molecule chaperone inhibitor, activates the heat shock response in Arabidopsis. Although sanguinarine inhibited the chaperone activities of wheat germ extract much like geldanamycin, berberine and papaverine influenced the activities very little. These results suggest that sanguinarine may promote the heat shock response by regulating the chaperone activities in the way that geldanamycin does in plants.

Glucosinolate metabolism, functionality and breeding for the improvement of Brassicaceae vegetables.

Unique secondary metabolites, glucosinolates (S-glucopyranosyl thiohydroximates), are naturally occurring S-linked glucosides found mainly in Brassicaceae plants. They are enzymatically hydrolyzed to produce sulfate ions, D-glucose, and characteristic degradation products such as isothiocyanates. The functions of glucosinolates in the plants remain unclear, but isothiocyanates possessing a pungent or irritating taste and odor might be associated with plant defense from microbes. Isothiocyanates have been studied extensively in experimental in vitro and in vivo carcinogenesis models for their cancer chemopreventive properties. The beneficial isothiocyanates, glucosinolates that are functional for supporting human health, have received attention from many scientists studying plant breeding, plant physiology, plant genetics, and food functionality. This review presents a summary of recent topics related with glucosinolates in the Brassica family, along with a summary of the chemicals, metabolism, and genes of glucosinolates in Brassicaceae. The bioavailabilities of isothiocyanates from certain functional glucosinolates and the importance of breeding will be described with emphasis on glucosinolates.

Precise, fast and flexible determination of protein interactions by affinity capillary electrophoresis. Part 2: cations.

The influence of various cations as metal ions (barium, calcium, copper, magnesia, manganese, and nickel), pharmaceuticals (ephedrine, ethambutol, pilocarpine, and pirenzepine), arginine, and guanidine has been tested on BSA, ß-lactoglobulin, and ovalbumin. Influences on proteins regarding changes in size, charge, or mass were of particular interest. ACE proved to be a suitable method to investigate these effects. ACE is able to observe slight but significant changes on proteins due to the excellent precision of the measurements. Therefore, some unexpected behaviors of protein-ligand interactions were found. The protein charge becomes more negative under metal ion influence and some pharmaceutical cations. Probably metal ions bound to the proteins form additional complexes with anions from the surrounding solution. Furthermore, already bound cations could be displaced at the protein surface. Both effects would change the overall charge of the ligand-protein complexes. In all studied cases, multiple-binding stoichiometries have been observed.

A KS-type dehydrin and its related domains reduce Cu-promoted radical generation and the histidine residues contribute to the radical-reducing activities.

Dehydrin is a plant disordered protein whose functions are not yet totally understood. Here it is reported that a KS-type dehydrin can reduce the formation of reactive oxygen species (ROS) from Cu. AtHIRD11, which is the Arabidopsis KS-type dehydrin, inhibited generation of hydrogen peroxide and hydroxyl radicals in the Cu-ascorbate system. The radical-reducing activity of AtHIRD11 was stronger than those of radical-silencing peptides such as glutathione and serum albumin. The addition of Cu(2+) reduced the disordered state, decreased the trypsin susceptibility, and promoted the self-association of AtHIRD11. Domain analyses indicated that the five domains containing histidine showed ROS-reducing activities. Histidine/alanine substitutions indicated that histidine is a crucial residue for reducing ROS generation. Using the 27 peptides which are related to the KnS-type dehydrins of 14 plant species, it was found that the strengths of ROS-reducing activities can be determined by two factors, namely the histidine contents and the length of the peptides. The degree of ROS-reducing activities of a dehydrin can be predicted using these indices.

Functional characterization of aromatic amino acid aminotransferase involved in 2-phenylethanol biosynthesis in isolated rose petal protoplasts.

In rose flowers, 2-phenylethanol (2PE) is biosynthesized from l-phenylalanine (l-Phe) via phenylacetaldehyde (PAld) by the actions of two enzymes, pyridoxal-5'-phosphate (PLP)-dependent aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). We here report that Rosa 'Yves Piaget' aromatic amino acid aminotransferase produced phenylpyruvic acid (PPA) from l-Phe in isolated petal protoplasts. We have cloned three full length cDNAs (RyAAAT1-3) of aromatic amino acid aminotransferase families based on rose EST database and homology regions. The RyAAATs enzymes were heterogeneously expressed in Escherichia coli and characterized biochemically. The recombinant RyAAAT3 showed the highest activity toward l-Phe in comparison with l-tryptophan, l-tyrosine, d-Phe, glycine, and l-alanine, and showed 9.7-fold higher activity with l-Phe rather than PPA as a substrate. RyAAAT3 had an optimal activity at pH 9 and at 45-55°C with α-ketoglutaric acid, and was found to be a PLP dependent enzyme based on the inhibition test using Carbidopa, an inhibitor of PLP-dependent enzymes. The transcript of RyAAAT3 was expressed in flowers as well as other organs of R. 'Yves Piaget'. RNAi suppression of RyAAAT3 decreased 2PE production, revealing the involvement of RyAAAT3 in 2PE biosynthesis in rose protoplasts and indicating that rose protoplasts have potentially two different 2PE biosynthetic pathways, the AADC route and the new route via PPA from l-Phe.

The multifunctionality of dehydrins: an overview.

Dehydrins are highly hydrophilic proteins that accumulate during embryogenesis and water stress responses in plants. Although dehydrins were discovered in the 1980s, their physiological functions are unknown. However, recent molecular-based studies have provided insights into the multifunctionality of dehydrins. The functional versatility of dehydrins is reviewed using recent experimental evidence, and perspectives in the functional studies of dehydrins are also discussed.

Exogenously applied isothiocyanates enhance glutathione S-transferase expression in Arabidopsis but act as herbicides at higher concentrations.

Isothiocyanates (ITCs) are sulfur-containing compounds that are generated by the glucosinolate-myrosinase system in plants. Although previous greenhouse studies have demonstrated the phytotoxicity of ITCs, their action modes are still unknown. In this study, we report the physiological responses of Arabidopsis thaliana treated with three exogenous ITCs: methyl ITC, allyl ITC, and phenethyl ITC. Administration of a high dose of each ITC inhibited plant growth and induced severe bleaching in the rosette leaves. The bleaching was concomitant with the elevation of electrolyte leakage and the generation of hydrogen peroxide. Although the three ITCs showed bleaching symptoms, phenethyl ITC was the most potent. A low dose of phenethyl ITC, at which the ITC did not promote leaf bleaching, enhanced the accumulation of transcripts of glutathione S-transferases (GSTs) in Arabidopsis. When 16 GST genes were tested, the levels of transcripts corresponding to 5 of the GST genes were enhanced in response to the phenethyl ITC treatment. In particular, the expression of a Tau class gene (AtGSTU19, At1g78380) responded to the phenethyl ITC treatment. Enhancement of the AtGSTU19 gene expression also occurred in the treatment of both allyl ITC and methyl ITC. These results suggest that the administration of ITCs to Arabidopsis at high doses has an herbicidal effect by inducing oxidative burst-like responses, but that administration at lower doses enhances the expression of specific GST genes in Arabidopsis.