Glutamine (Gln), the first organic nitrogen (N) produced during primary N assimilation, is increasingly recognised as a signalling molecule in plants. Here, we show that Gln, supplied as the sole N source, promotes root hair elongation in Arabidopsis seedlings compared with NH4NO3. This response is dose-dependent and persists even under high NH4NO3 concentrations. Transcriptomic and gene expression analyses showed that Gln represses the negative regulator GLABRA2 while inducing the positive regulators ROOT HAIR DEFECTIVE6 (RHD6), RHD6-LIKE2, and RHD6-LIKE4, which control root hair development. Consistently, the rhd6-1 mutant failed to develop root hairs in response to Gln, demonstrating that Gln-induced root hair elongation depends on the RHD6 pathway. Gln also induces defence gene expression. Our pharmacological studies revealed that proper pectin methyl-esterification is required for both Gln-induced root hair elongation and defence gene expression, highlighting the importance of cell wall integrity in perceiving exogenous Gln. Furthermore, we show that Gln perception at the root apex triggers root hair elongation-a response that relies on defence hormone signalling, particularly ethylene, as demonstrated by pharmacological and genetic analyses. Together, these findings suggest that Gln integrates nutrient sensing, cell wall integrity, and defence hormone signalling to regulate root hair elongation in Arabidopsis.

ICE1 (Inducer of CBF Expression 1) of Arabidopsis thaliana is a transcription factor involved in a wide range of physiological processes, including stress responses and plant development. Recently, the regulatory role of ICE1 in the development of the male reproduction system was reported; the failure of the ICE1 mutant ice1-2 to carry out anther dehydration was observed to have resulted from impaired stomata development. In this study, we show that ICE1 influences anther and pollen development by facilitating timely tapetum degradation and nutrient mobilization. Transmission electron microscopy revealed that ice1-2 pollen grains exhibited disorganized cytoplasm and large vacuoles. Semi-thin sectioning of ice1-2 anthers from different developmental stages showed retarded anther dehiscence, defective pollen grains, and delayed tapetum degradation. In addition, ice1-2 exhibited altered sugar and lipid distribution patterns, suggesting that abnormal pollenkitt deposition results from impaired nutrient mobilization and defective tapetal degradation. Lastly, we also investigated and confirmed interactions between ICE1 and key regulators of tapetum and pollen development, DYT1 and AMS. Together, our observations suggest an additional role of ICE1 in anther and pollen development via the regulation of tapetum development, likely through the interactions of ICE1 and other transcription factors involved in tapetal and pollen development.

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 phospho-transferase 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 hy-droxyl 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.

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.

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.

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 (PrP C ) into the misfolded, β-sheet-rich and aggregation-prone isoform (PrP Sc ), 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 PrP C ) and 200–227 (helix 3 in PrP C ) 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.

The divergence of duplicate genes links to organismic adaptation. In Arabidopsis thaliana two nuclear genes encode mitochondrial GrpEs, MGE1 and MGE2, the nucleotide exchange factors of DnaK/HSP70 chaperone. MGE1 and MGE2 are duplicate genes originated from a whole genome duplication event. They respond differentially to high temperature; MGE2 is heat-inducible and is required for Arabidopsis seedlings to tolerate prolonged heat stress, while MGE1 is constitutively expressed. Heterologous expression of MGE2 but not MGE1 restored the growth of E. coli grpE mutant cells at elevated temperatures, suggesting that MGE2 is more thermostable than MGE1. In this study, we directly compared the thermostability of the purified recombinant MGE1 and MGE2 by circular dichroism spectroscopy. The temperature midpoints of the unfolding transition (Tm) of MGE1 and MGE2 were about 38 and 46 °C, respectively, indicating that MGE2 is remarkably more stable than MGE1 at higher temperature. Domain swapping between the two homologous proteins showed that the N-terminal region, including an unstructured sequence and a long α-helix domain, is the major determinant of the thermostability. Although MGE2 contains a conserved sequence derived from an exonized intron within the N-terminus unstructured region, deletion of this sequence did not substantially affect protein thermostability in vitro and complementation of E. coli and Arabidopsis heat sensitive mutants. Taken together, our results suggest that Arabidopsis MGE1 and MGE2 had diverged not only in transcriptional response but also in the thermostability of the encoded proteins, which may contribute to adaptation of plants to higher temperatures.

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-3β-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.

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.

An ethylene receptor gene named BoERS1 was cloned from a bamboo (Bambusa oldhamii) cDNA library. The open reading frame of BoERS1 was 1,899 bp and encoded a 632-amino acid protein, which contains the five conserved motifs (H, N, G1, F, and G2 boxes) of the bacterial two-component system histidine kinases and shows high sequence similarity with other ethylene receptors in plants, such as rice and maize. Expression of BoERS1 in bamboo shoots increased with the growth of the emerging shoots. In an in vitro kinase assay, the expressed histidine kinase domain of BoERS1 (BHK) was phosphorylated in the presence of Mn2+, and LC-ESI-MS/MS analysis showed that four amino acids, namely T442, S444, S489, and S503, were phosphorylated. It is interesting to note that S489 and S503 are located in a loop region (L1) that is found only in plant histidine kinase-containing enzymes. The identification of multiple phosphorylation sites on BoERS1 provides a new avenue for future structure–function studies of the ethylene receptor protein family.

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.

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.

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 β-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).

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.

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 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 β-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.

Cytokinins (CKs) are a group of phytohormones involved in stages of plant development, such as cell division and various other growth processes. The key enzyme in de 1101;0 biosynthesis of naturally occurring cytokinins is adenylate isopentenyltransferase (AIPT). This study amplified a PCR product, containing partial sequence encoding putative AIPT, using primers based on conserved protein sequences. Genomic DNA containing a full-length of AIPT was obtained by screening a genomic DNA library. This study used 5'-RACE and 3'-RACE, using gene specific primers, to clone and sequence cDNA encoding BoAIPTI. The open reading frame of BoAIPTI was 1035 bp in size, encoding 344-amino acids, with a molecular mass of 37.7 kDa. No introns were found by comparing AIPT from bamboo with AIPT sequences from other species. BoAIPTI contains a putative ATP/GTP binding motif at the N-terminal as observed in other known AIPT. Recombinant BoAIPTI expressed in Escherichia coli catalyzed the isopentenyl transfer reaction from dimethylallyldiphosphate (DMAPP) to the N-6 adenylate amino group. Real-time PCR analyses revealed BoAIPTI expression increased simultaneously with green bamboo shoot growth, with the top green shoot section having the highest expression level.

Amyloidosis is a rare disease characterized by the deposition of abnormal proteins in extracellular tissues. We herein report a case with instructive radiologic features of nodular pulmonary amyloidosis associated with Sjögren's syndrome. A 67-year-old woman was referred to our department because of an abnormal chest radiograph. Chest computed tomography revealed multiple round cysts accompanied by calcified nodules. The patient was clinically diagnosed with primary Sjögren's syndrome and pathologically diagnosed with nodular pulmonary amyloidosis (light chain, kappa). Although multiple lung cysts have many etiologies, the presence of calcified nodules associated with multiple lung cysts is useful for narrowing down the differential diagnosis.

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.