Discovery of a novel phosphotransferase from Bacillus subtilis that phosphorylates a broad spectrum of flavonoids.

Flavonoids are associated with health benefits, but most of them have poor oral bioavailability due to their extremely low aqueous solubility. Flavonoid O-phosphorylation suggests a potent modification to solve the problems. Here, we isolated, identified and characterized an unprecedented phosphotransferase, flavonoid phosphate synthetase (BsFPS), from B. subtilis. The enzyme catalyzes the ATP-dependent phosphorylation of flavonoid to generate flavonoid monophosphates, AMP and orthophosphate. BsFPS is a promiscuous phosphotransferase that efficiently catalyzes structurally-diverse flavonoids, including isoflavones, flavones, flavonols, flavanones and flavonolignans. Based on MS and NMR analysis, the phosphorylation mainly occurs on the hydroxyl group at C-7 of A-ring or C-4' of B-ring in flavonoid skeleton. Notably, BsFPS is regioselective for the ortho-3',4'-dihydroxy moiety of catechol-containing structures, such as luteolin and quercetin, to produce phosphate conjugates at C-4' or C-3' of B-ring. Our findings highlight the potential for developing biosynthetic platform to obtain new phosphorylated flavonoids for pharmaceutical and nutraceutical applications.

Nitrogen deficiency- and sucrose-induced anthocyanin biosynthesis is modulated by HISTONE DEACETYLASE15 in Arabidopsis.

Anthocyanin accumulation is a hallmark response to nitrogen (N) deficiency in Arabidopsis. Although the regulation of anthocyanin biosynthesis has been extensively studied, the roles of chromatin modification in this process are largely unknown. In this study we show that anthocyanin accumulation induced by N deficiency is modulated by HISTONE DEACETYLASE15 (HDA15) in Arabidopsis seedlings. The hda15-1 T-DNA insertion mutant accumulated more anthocyanins than the wild-type when the N supply was limited, and this was caused by up-regulation of anthocyanin biosynthetic and regulatory genes in the mutant. The up-regulated genes also had increased levels of histone acetylation in the mutant. The accumulation of anthocyanins induced by sucrose and methyl jasmonate, but not that induced by H2O2 and phosphate starvation, was also greater in the hda15-1 mutant. While sucrose increased histone acetylation in the hda15-1 mutant in genes in a similar manner to that caused by N deficiency, methyl jasmonate only enhanced histone acetylation in the genes involved in anthocyanin biosynthesis. Our results suggest that different stresses act through distinct regulatory modules to activate anthocyanin biosynthesis, and that HDA15-mediated histone modification modulates the expression of anthocyanin biosynthetic and regulatory genes to avoid overaccumulation in response to N deficiency and other stresses.

De Novo Design of Antimicrobial Peptides With a Special Charge Pattern and Their Application in Combating Plant Pathogens.

Plant diseases are important issues in agriculture, and the development of effective and environment-friendly means of disease control is crucial and highly desired. Antimicrobial peptides (AMPs) are known as potential alternatives to chemical pesticides because of their potent broad-spectrum antimicrobial activity and because they have no risk, or have only a low risk, of developing chemical-resistant pathogens. In this study, we designed a series of amphipathic helical peptides with different spatial distributions of positive charges and found that the peptides that had a special sequence pattern "BBHBBHHBBH" ("B" for basic residue and "H" for hydrophobic residue) displayed excellent bactericidal and fungicidal activities in a wide range of economically important plant pathogens. The peptides with higher helical propensity had lower antimicrobial activity. When we modified the peptides with a long acyl chain at their N-terminus, their plant protection effect improved. Our application of the fatty acyl-modified peptides on the leaves of tomato and Arabidopsis plants lessened the infection caused by Pectobacterium carotovorum subsp. carotovorum and Botrytis cinerea. Our study provides important insights on the development of more potent novel AMPs for plant protection.

Segments in the Amyloid Core that Distinguish Hamster from Mouse Prion Fibrils.

Prion diseases are transmissible fatal neurodegenerative disorders affecting humans and other mammals. The disease transmission can occur between different species but is limited by the sequence homology between host and inoculum. The crucial molecular event in the progression of this disease is prion formation, starting from the conformational conversion of the normal, membrane-anchored prion protein (PrPC) into the misfolded, ß-sheet-rich and aggregation-prone isoform (PrPSc), which then self-associates into the infectious amyloid form called prion. Amyloid is the aggregate formed from one-dimensional protein association. As amyloid formation is a key hallmark in prion pathogenesis, studying which segments in prion protein are involved in the amyloid formation can provide molecular details in the cross-species transmission barrier of prion diseases. However, due to the difficulties of studying protein aggregates, very limited knowledge about prion structure or prion formation was disclosed by now. In this study, cross-seeding assay was used to identify the segments involved in the amyloid fibril formation of full-length hamster prion protein, SHaPrP(23-231). Our results showed that the residues in the segments 108-127, 172-194 (helix 2 in PrPC) and 200-227 (helix 3 in PrPC) are in the amyloid core of hamster prion fibrils. The segment 127-143, but not 107-126 (which corresponds to hamster sequence 108-127), was previously reported to be involved in the amyloid core of full-length mouse prion fibrils. Our results indicate that hamster prion protein and mouse prion protein use different segments to form the amyloid core in amyloidogenesis. The sequence-dependent core formation can be used to explain the seeding barrier between mouse and hamster.

Antcins, triterpenoids from Antrodia cinnamomea, as new agonists for peroxisome proliferator-activated receptor α.

Peroxisome proliferator-activated receptor α (PPARα) is a nuclear hormone receptor that transcriptionally regulates lipid metabolism and inflammation; therefore, PPARα agonists are promising agents to treat dyslipidemia and metabolic disorders. PPARα full agonists, such as fibrates, are effective anti-hypertriglyceride agents, but their use is limited by adverse side effects. Hence, the aim of this study was to identify small molecules that can activate PPARα while minimizing the adverse effects. Antrodia cinnamomea, a rare medical mushroom, has been used widely in Asian countries for the treatment of various diseases, including liver diseases. Antcin B, H and K (antcins) and ergostatrien-3ß-ol (EK100) are bioactive compounds isolated from A. cinnamomea with anti-inflammatory actions. Antcins, ergostane-type triterpenoids, contain the polar head with carboxylate group and the sterol-based body. Here, we showed at the first time that sterol-based compounds, antcins, but not EK100, activate PPARα in a cell-based transactivation study. The in silico docking studies presented several significant molecular interactions of antcins, including Tyr314, and His440 in the ligand-binding domain of PPARα, and these interactions are required for helix 12 (H12) stabilization. We propose that PPARα activation activity of antcins is related to their binding mode which requires conventional H12 stabilization, and that antcins can be developed as safe selective PPARα modulators.

An ultra-compact blackbody using electrophoretic deposited carbon nanotube films.

Carbon nanotubes (CNTs) possesses decent optical properties and thus can be considered as a candidate for perfect absorbers due to their close-to-air refractive index and minimal extinction. However, weak absorption in porous materials, due to the low extinction coefficients, requires an inevitably thick absorption layer (∼100 µm) for the perfect opaque absorbers. Thus, the requirement of large thicknesses of CNTs prohibits them from being used as miniaturized integrated photonic devices. Here, we propose an electrophoretic deposited (EPD) CNT resonant cavity structure on tantalum (Ta) to enhance optical absorption. Efficient random light scattering along with the resonant cavity structure using Ti/SiO2 stacking enhances the absorption in our proposed EPD-CNT film while maintaining the total device thickness to <1 µm. The experiment results reveal that the absorption band covers the entire UV-VIS-NIR spectrum (λ = 0.3-2.6 µm), using resonant-cavity EPD-CNT design. The EPD deposition process is done at relatively low temperature < 120 °C. We believe that this proposal is very promising for sensing, antenna, and thermophotovoltaics (TPV), in terms of bandwidth, compactness and cost.

New insight into the catalytic properties of rice sucrose synthase.

Sucrose synthase (SuS), which catalyzes the reversible conversion of sucrose and uridine diphosphate (UDP) into fructose and UDP-glucose, is a key enzyme in sucrose metabolism in higher plants. SuS belongs to family 4 of the glycosyltransferases (GT4) and contains an E-X7-E motif that is conserved in members of GT4 and two other GT families. To gain insight into the roles of this motif in rice sucrose synthase 3 (RSuS3), the two conserved glutamate residues (E678 and E686) in this motif and a phenylalanine residue (F680) that resides between the two glutamate residues were changed by site-directed mutagenesis. All mutant proteins maintained their tetrameric conformation. The mutants E686D and F680Y retained partial enzymatic activity and the mutants E678D, E678Q, F680S, and E686Q were inactive. Substrate binding assays indicated that UDP and fructose, respectively, were the leading substrates in the sucrose degradation and synthesis reactions of RSuS3. Mutations on E678, F680, and E686 affected the binding of fructose, but not of UDP. The results indicated that E678, F680, and E686 in the E-X7-E motif of RSuS3 are essential for the activity of the enzyme and the sequential binding of substrates. The sequential binding of the substrates implied that the reaction catalyzed by RSuS can be controlled by the availability of fructose and UDP, depending on the metabolic status of a tissue.

Identification of ICE1 as a negative regulator of ABA-dependent pathways in seeds and seedlings of Arabidopsis.

Inducer of CBF expression 1 (ICE1) mediates the cold stress signal via an abscisic acid (ABA)-independent pathway. A possible role of ICE1 in ABA-dependent pathways was examined in this study. Seedling growth was severely reduced in a T-DNA insertion mutant of ICE1, ice1-2, when grown on 1/2 MS medium lacking sugars, but was restored to wild-type (WT) levels by supplementation with 56 mM glucose. In addition to this sugar-dependent phenotype, germination and establishment of ice1-2 were more sensitive to high glucose concentrations than in the WT. Hypersensitivity to ABA was also observed in ice1-2, suggesting its sensitivity to glucose might be mediated through the ABA signaling pathway. Glucose and ABA induced much higher expression of two genes related to ABA signal transduction, ABA-INSENSITIVE 3 (ABI3) and ABA-INSENSITIVE 4 (ABI4), in ice1-2 than in the WT during establishment. In summary, in addition to its known roles in regulating cold responses, stomatal development, and endosperm breakdown, ICE1 is a negative regulator of ABA-dependent responses.

Tentative identification of the second substrate binding site in Arabidopsis phytochelatin synthase.

Phytochelatin synthase (PCS) uses the substrates glutathione (GSH, γGlu-Cys-Gly) and a cadmium (Cd)-bound GSH (Cd∙GS2) to produce the shortest phytochelatin product (PC2, (γGlu-Cys)2-Gly) through a ping-pong mechanism. The binding of the 2 substrates to the active site, particularly the second substrate binding site, is not well-understood. In this study, we generated a structural model of the catalytic domain of Arabidopsis AtPCS1 (residues 12-218) by using the crystal structure of the γGlu-Cys acyl-enzyme complex of the PCS of the cyanobacterium Nostoc (NsPCS) as a template. The modeled AtPCS1 revealed a cavity in proximity to the first substrate binding site, consisting of 3 loops containing several conserved amino acids including Arg152, Lys185, and Tyr55. Substitutions of these amino acids (R152K, K185R, or double mutation) resulted in the abrogation of enzyme activity, indicating that the arrangement of these 2 positive charges is crucial for the binding of the second substrate. Recombinant AtPCS1s with mutations at Tyr55 showed lower catalytic activities because of reduced affinity (3-fold for Y55W) for the Cd∙GS2, further suggesting the role of the cation-π interaction in recognition of the second substrate. Our study results indicate the mechanism for second substrate recognition in PCS. The integrated catalytic mechanism of PCS is further discussed.

Amyloid core formed of full-length recombinant mouse prion protein involves sequence 127-143 but not sequence 107-126.

The principal event underlying the development of prion disease is the conversion of soluble cellular prion protein (PrP(C)) into its disease-causing isoform, PrP(Sc). This conversion is associated with a marked change in secondary structure from predominantly α-helical to a high ß-sheet content, ultimately leading to the formation of aggregates consisting of ordered fibrillar assemblies referred to as amyloid. In vitro, recombinant prion proteins and short prion peptides from various species have been shown to form amyloid under various conditions and it has been proposed that, theoretically, any protein and peptide could form amyloid under appropriate conditions. To identify the peptide segment involved in the amyloid core formed from recombinant full-length mouse prion protein mPrP(23-230), we carried out seed-induced amyloid formation from recombinant prion protein in the presence of seeds generated from the short prion peptides mPrP(107-143), mPrP(107-126), and mPrP(127-143). Our results showed that the amyloid fibrils formed from mPrP(107-143) and mPrP(127-143), but not those formed from mPrP(107-126), were able to seed the amyloidogenesis of mPrP(23-230), showing that the segment residing in sequence 127-143 was used to form the amyloid core in the fibrillization of mPrP(23-230).

Identification of genes differentially expressed during the growth of Bambusa oldhamii.

Bamboos are ecologically and economically important grasses, and are distinguished by their rapid growth. To identify genes associated with bamboo growth, PCR-based mRNA differential display was used to clone genes that were differentially expressed in various tissues of bamboo (Bambusa oldhamii) shoots at different growth stages. In total, 260 different cDNA sequences were obtained. These genes displayed complex expression profiles across the different tissues and growth stages as revealed by a cDNA microarray analysis. Notable among them were genes that were temporally up-regulated or down-regulated in the internode-containing region of rapidly elongating shoots. These genes might participate in the rapid elongation of the bamboo culm. Of the 36 up-regulated and 46 down-regulated genes, 16 genes and 8 genes, respectively, were predicted to encode hypothetical proteins or were unknown sequences. Aside from these, genes involved in hormonal signaling and homeostasis, stress responses, peptide processing and signaling and lignin biosynthesis composed most of the up-regulated genes; genes involved in DNA replication, nucleic acid binding and signal transduction were highly represented among the down-regulated genes. These results suggested that genes associated with plant hormonal signaling and homeostasis, peptide signaling, reactive oxygen species signaling and homeostasis, several stress-related genes and a monocot-specific unknown gene, BoMSP41, play important roles in the elongation of bamboo internodes. Multiple signaling pathways might form a complex interconnected network that controls the rapid growth of this giant grass.

E3-independent ubiquitination of AtMAPR/MSBP1.

AtMAPR5/MSBP1 and its homologs can be ubiquitinated in the absence of E3 ligase in in vitro ubiquitination assays. Ubiquitinated AtMAPR3, AtMAPR5/MSBP1, and AtMAPR2 were identified using LC-MS/MS. Analysis of trypsin-released signature peptides showed that this E3-independent ubiquitination of AtMAPR3, AtMAPR5/MSBP1, and AtMAPR2 was dominated by mono-ubiquitination at multiple sites. Unlike AtUBC8-type E2s, AtUBC36 was not able to transfer ubiquitin to AtMAPR2. The truncated mutants AtMAPR2Δ1-10, AtMAPR2Δ1-30, and AtMAPR2_1-73 could also be ubiquitinated. The presence of a ubiquitin-binding domain (UBD) allows proteins to be ubiquitinated independently of E3 ligases. However, AtMAPRs do not contain any known UBD. In vitro ubiquitination of AtMAPR2 observed in this study will be further studied in biochemical and physiological aspects.

The purine-rich DNA-binding protein OsPurα participates in the regulation of the rice sucrose synthase 1 gene expression.

The rice sucrose synthase 1 (RSus1) gene is transcriptionally induced by sucrose, and a region within its promoter, at -1117 to -958 upstream of the transcription initiation site, was found to be essential for enhancing the sucrose-induced expression. Further dissection of this region revealed that a group of nuclear proteins interact with a 39-bp fragment named A-3-2 (-1045 to -1007). A protein that specifically and directly interacted with A-3-2 was isolated from the suspension-cultured cells of rice and was subsequently identified as a purine-rich DNA-binding protein. The amino acid sequence of this protein, OsPurα, exhibited 73% identity with the Arabidopsis Purα-1 protein, and its modeled structure resembled the structure of Pur-α in Drosophila. Recombinant OsPurα expressed and purified from Escherichia coli was demonstrated to have DNA-binding activity and to interact with A-3-2 specifically. Moreover, OsPurα was able to enhance sucrose-induced expression of the ß-glucuronidase (GUS) reporter gene, which was transcriptionally fused to two copies of a DNA fragment containing A-3-2 and the cauliflower mosaic virus 35S minimal promoter, in vivo. The level of OsPurα bound to A-3-2 was higher in cells cultured in the presence of sucrose; however, the level of OsPurα mRNA in cells was not affected by sucrose. The results of this study demonstrate that OsPurα participates in the regulation of RSus1 expression in response to sucrose; nevertheless, it may require other partner proteins for full function.

Molecular characterization of a phenylalanine ammonia-lyase gene (BoPAL1) from Bambusa oldhamii.

Phenylalanine ammonia-lyase is the first enzyme of general phenylpropanoid pathway. A PAL gene, designated as BoPAL1, was cloned from a Bambusa oldhamii cDNA library. The open reading frame of BoPAL1 was 2,139 bp in size and predicted to encode a 712-amino acid polypeptide. BoPAL1 was the first intronless PAL gene found in angiosperm plant. Several putative cis-acting elements such as P box, GT-1motif, and SOLIPs involved in light responsiveness were found in the 5'-flanking sequence of BoPAL1 which was obtained by TAIL-PCR method. Recombinant BoPAL1 protein expressed in Pichia pastoris was active. The optimum temperature and pH for BoPAL1 activity was 50°C and 9.0, respectively. The molecular mass of recombinant BoPAL1 was estimated as 323 kDa using gel filtration chromatography and the molecular mass of full-length BoPAL was about 80 kDa, indicating that BoPAL1 presents as a homotetramer. The Km and kcat values of BoPAL1 for L-Phe were 1.01 mM and 10.11 s(-1), respectively. The recombinant protein had similar biochemical properties with PALs reported in other plants.

Cloning, expression, site-directed mutagenesis and immunolocalization of phenylalanine ammonia-lyase in Bambusa oldhamii.

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) from green bamboo was isolated and cloned from the shell of Bambusa oldhamii. The K(m) of bamboo shell PAL for L-Phe was 476 µM, and the molecular mass of native PAL was estimated as 275 kDa and the molecular mass of a subunit was about 76 kDa, indicating that PAL from bamboo also exists as a tetramer. The optimum temperature for PAL activity was 50°C and the optimal pH 9.0. The identity of the purified bamboo shell PAL was confirmed using Q-TOF tandem MS/MS de novo sequencing. Four PAL genes, designated as BoPAL1 to BoPAL4, were cloned from B. oldhamii. The open reading frames of BoPAL3 and BoPAL4 were 2142 and 2106 bp in size, respectively: BoPAL2-4 contained one intron and two exons, but no intron was found in BoPAL1. BoPAL4 expressed in Escherichia coli possessed both PAL and tyrosine ammonia-lyase activities. While recombinant wild-type PAL proteins had similar biochemical properties to the native bamboo shell PAL, both site-directed mutagenesis of BoPAL1 F133H and BoPAL2 F134H, respectively, showed decreased k(cat)/K(m) values toward L-Phe, whereas BoPAL2 F134H showed a slightly increased k(cat)/K(m) value toward L-Tyr. These data suggest other residues largely control Phe/Tyr substrate specificity. An antibody raised against the purified shell PAL was generated for histochemical studies. In bamboo shell and branch shoots, PAL was localized primarily in sclerenchyma cells.

Analysis of the cellulose synthase genes associated with primary cell wall synthesis in Bambusa oldhamii.

The synthesis of cell wall polysaccharides is highly active in rapidly growing bamboo shoots. We cloned a set of BoCesA cDNAs that encode cellulose synthase from bamboo (Bambusa oldhamii) and investigated the expression patterns of the BoCesA2, BoCesA5, BoCesA6 and BoCesA7 genes. The four BoCesA genes were differentially expressed in the different parts of growing bamboo shoots, in various organs, and in multiple shoots that were cultured in vitro. They were down-regulated by alpha-naphthaleneacetic acid and differentially affected by thidiazuron in the multiple shoots. In situ RT-PCR analyses demonstrated that BoCesA2, BoCesA5, BoCesA6, and BoCesA7 mRNAs were present throughout the base and the internode regions of the etiolated shoots that emerged from pseudorhizomes, and in the internode regions of the juvenile branch shoots that emerged from nodes of mature bamboo culms; however, the expression of the four genes in the lignified internode of the branch shoot was predominantly detected in the center of the vascular bundles. Our results for cDNA cloning, expression analyses, and phylogenetic analysis suggest that the 10 BoCesA genes cloned from the etiolated bamboo shoots participate in cellulose synthesis in the primary cell walls of the growing bamboo, and that at least three additional BoCesA genes involved in cellulose synthesis in the secondary walls may be present in the bamboo genome. The expressions of BoCesA genes may be under fine control in response to the various developmental stages and physiological conditions of bamboo.

HHP1, a novel signalling component in the cross-talk between the cold and osmotic signalling pathways in Arabidopsis.

Heptahelical protein 1 (HHP1) is a negative regulator in abscisic acid (ABA) and osmotic signalling in Arabidopsis. The physiological role of HHP1 was further investigated in this study using transgenic and knock-out plants. In HHP1::GUS transgenic mutants, GUS activity was found to be mainly expressed in the roots, vasculature, stomata, hydathodes, adhesion zones, and connection sites between septa and seeds, regions in which the regulation of turgor pressure is crucial. By measuring transpiration rate and stomatal closure, it was shown that the guard cells in the hhp1-1 mutant had a decreased sensitivity to drought and ABA stress compared with the WT or the c-hhp1-1 mutant, a complementation mutant of HHP1 expressing the HHP1 gene. The N-terminal fragment (amino acids 1-96) of HHP1 was found to interact with the transcription factor inducer of CBF expression-1 (ICE1) in yeast two-hybrid and bimolecular fluorescence complementation (BiFC) studies. The hhp1-1 mutant grown in soil showed hypersensitivity to cold stress with limited watering. The expression of two ICE1-regulated genes (CBF3 and MYB15) and several other cold stress-responsive genes (RD29A, KIN1, COR15A, and COR47) was less sensitive to cold stress in the hhp1-1 mutant than in the WT. These data suggest that HHP1 may function in the cross-talk between cold and osmotic signalling.

Cloning and expression of a phenylalanine ammonia-lyase gene (BoPAL2) from Bambusa oldhamii in Escherichia coli and Pichia pastoris.

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is the first committed enzyme of phenylpropanoid pathway. A PAL gene, designated as BoPAL2, was cloned from a Bambusa oldhamii cDNA library. The open reading frame of BoPAL2 was 2142bp in size encoding a 713-amino acid polypeptide. BoPAL2 was heterologous expressed in Escherichia coli and Pichia pastoris. The recombinant proteins were exhibited PAL and tyrosine ammonia-lyase activities. The recombinant BoPAL2 had a subunit mass of 80kDa and existed as a homotetramer. The optimum temperature and pH of BoPAL2 were 50-60 degrees C and 8.5-9.0, respectively. The K(m) and k(cat) values of BoPAL2 expressed in E. coli were 250microM and 10.12s(-1). The K(m) and k(cat) values of BoPAL2 expressed in P. pastoris were 331microM and 16.04s(-1). The recombinant proteins had similar biochemical properties and kinetic parameters with PALs reported in other plants.

Phytochelatin synthase is regulated by protein phosphorylation at a threonine residue near its catalytic site.

Heavy metals are toxic to most living organisms and cause health problems by contaminating agricultural products. In plants, phytochelatin synthase (PCS, EC 2.3.2.15) uses glutathione (GSH) as its substrate to catalyze the synthesis of heavy metal-binding peptides, known as phytochelatins (PC). PCS has been described as a constitutive enzyme that may be controlled by post-translational modifications. However, the detailed mechanism of its catalytic activity is not clear. In this study, in vitro experiments demonstrate that PCS activity increased following phosphorylation by casein kinase 2 (CK2) and decreased following treatment with alkaline phosphatase. Site-directed mutagenesis experiments at amino acids on AtPCS1 indicate that Thr 49 is the site for phosphorylation. This is further supported by fact that the mutant AtPCS1(T49A) cannot be phosphorylated, and its activity is significantly lower than that of the wild-type enzyme. In the modeled three-dimensional structure of AtPCS1, Arg 183 is within close proximity to Thr 49. The mutant AtPCS1(R183A) can be phosphorylated, but it shows much lower catalytic activity than the wild-type protein. This result suggested that Arg 183 may play an important role in the catalytic mechanism of AtPCS1. The possibility of the presence of a second substrate-binding site as a result of the interaction of these two amino acids is discussed. In addition, the activity of AtPCS1 was also found to be modulated by the C-terminal domain. The N-terminal catalytic domain of AtPCS1 was expressed (AtPCS1-N), and its catalytic activity was found to be even more sensitive to Cd or phosphorylation status than was the full-length enzyme.

HHP1 is involved in osmotic stress sensitivity in Arabidopsis.

HHP1 (heptahelical protein 1), a protein with a predicted seven transmembrane domain structure homologous to adiponectin receptors (AdipoRs) and membrane progestin receptors (mPRs), has been characterized. Expression of HHP1 was increased in response to abscisic acid (ABA) and salt/osmotic stress as shown by quantitative real-time PCR and HHP1 promoter-controlled GUS activity. The HHP1 T-DNA insertion mutant (hhp1-1) showed a higher sensitivity to ABA and osmotic stress than the wild-type (WT), as revealed by the germination rate and post-germination growth rate. The induced expression of stress-responsive genes (RD29A, RD29B, ADH1, KIN1, COR15A, and COR47) was more sensitive to exogenous ABA and osmotic stress in hhp1-1 than in the WT. The hypersensitivity in the hhp1-1 mutant was reversed in the complementation mutant of HHP1 expressing the HHP1 gene. The data suggest that the mutation of HHP1 renders plants hypersensitive to ABA and osmotic stress and HHP1 might be a negative regulator in ABA and osmotic signalling.