Role of Wisteria floribunda agglutinin binding glycans in carcinogenesis and metastasis of cholangiocarcinoma.

Aberrant glycosylation is an important factor in facilitating tumor progression and therapeutic resistance. In this study, using Wisteria floribunda agglutinin (WFA), we examined the expression of WFA-binding glycans (WFAG) in cholangiocarcinoma (CCA). The results showed that WFAG was highly detected in precancerous and cancerous lesions of human CCA tissues, although it was rarely detected in normal bile ducts. The positive signal of WFAG in the cancerous lesion accounted for 96.2% (50/52) of the cases. Overexpression of WFAG was significantly associated with lymph node and distant metastasis (P < 0.05). The study using the CCA hamster model showed that WFAG is elevated in preneoplastic and neoplastic bile ducts as early as 1 month after being infected with liver fluke and exposed to N-nitrosodimethylamine. Functional analysis was performed to reveal the role of WFAG in CCA. The CCA cell lines KKU-213A and KKU-213B were treated with WFA, followed by migration assay. Our data suggested that WFAG facilitates the migration of CCA cells via the activation of the Akt and ERK signaling pathways. In conclusion, we have demonstrated the association of WFAG with carcinogenesis and metastasis of CCA, suggesting its potential as a target for the treatment of the disease.

Establishment and characterization of a novel cancer stem-like cell of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive malignant tumor of bile duct epithelia. Recent evidence suggests the impact of cancer stem cells (CSC) on the therapeutic resistance of CCA; however, the knowledge of CSC in CCA is limited due to the lack of a CSC model. In this study, we successfully established a stable sphere-forming CCA stem-like cell, KKU-055-CSC, from the original CCA cell line, KKU-055. The KKU-055-CSC exhibits CSC characteristics, including: (1) the ability to grow stably and withstand continuous passage for a long period of culture in the stem cell medium, (2) high expression of stem cell markers, (3) low responsiveness to standard chemotherapy drugs, (4) multilineage differentiation, and (5) faster and constant expansive tumor formation in xenograft mouse models. To identify the CCA-CSC-associated pathway, we have undertaken a global proteomics and functional cluster/network analysis. Proteomics identified the 5925 proteins in total, and the significantly upregulated proteins in CSC compared with FCS-induced differentiated CSC and its parental cells were extracted. Network analysis revealed that high mobility group A1 (HMGA1) and Aurora A signaling through the signal transducer and activator of transcription 3 pathways were enriched in KKU-055-CSC. Knockdown of HMGA1 in KKU-055-CSC suppressed the expression of stem cell markers, induced the differentiation followed by cell proliferation, and enhanced sensitivity to chemotherapy drugs including Aurora A inhibitors. In silico analysis indicated that the expression of HMGA1 was correlated with Aurora A expressions and poor survival of CCA patients. In conclusion, we have established a unique CCA stem-like cell model and identified the HMGA1-Aurora A signaling as an important pathway for CSC-CCA.

Targeting alpha2,3-sialylated glycan in glioma stem-like cells by Maackia amurensis lectin-II: A promising strategy for glioma treatment.

Glioma stem/initiating cells have been considered a major cause of tumor recurrence and therapeutic resistance. In this study, we have established a new glioma stem-like cell (GSC), named U373-GSC, from the U373 glioma cell line. The cells exhibited stemness properties, e.g., expression of stem cell markers, self-renewal activity, multi-lineage differentiating abilities, and drug resistance. Using U373-GSC and GSC-03A-a GSC clone previously established from patient tissue, we have identified a novel GSC-associated sialic acid-modified glycan commonly expressed in both cell lines. Lectin fluorescence staining showed that Maackia amurensis lectin II (MAL-II)-binding alpha2,3-sialylated glycan (MAL-SG) was highly expressed in GSCs, and drastically decreased during FBS induced differentiation to glioma cells or little in the parental cells. Treatment of GSCs by MAL-II, compared with other lectins, showed that MAL-II significantly suppresses cell viability and sphere formation via induction of cell cycle arrest and apoptosis of the GSCs. Similar effects were observed when the cells were treated with a sialyltransferase inhibitor or sialidase. Taken together, we demonstrate for the first time that MAL-SGs/alpha-2,3 sialylations are upregulated and control survival/maintenances of GSCs, and their functional inhibitions lead to apoptosis of GSCs. MAL-SG could be a potential marker and therapeutic target of GSCs; its inhibitors, such as MAL-II, may be useful for glioma treatment in the future.

Anserine/Carnosine-Rich Extract from Thai Native Chicken Suppresses Melanogenesis via Activation of ERK Signaling Pathway.

Skin hyperpigmentation is an aesthetic problem that leads to psychosocial issues. Thus, skin whitening agents from agro- and poultry-industrial co-products are considered high economic value ingredients of interest for sustainable application. Therefore, this study aimed to determine the cosmeceutical potential of anserine/carnosine-rich chicken extract (ACCE) from the Thai native chicken Pradu Hang Dam Mor Kor 55 (PD) meat. The chemical composition was identified and quantified using the HPLC-UV method. Then, the antioxidation potential of the extract was compared to that of L-anserine and L-carnosine, using 1,1-diphenyl-2-picrylhydrazyl assay and shikonin-induced production of reactive oxygen species in CCD-986Sk cell models, and the anti-melanogenesis effect in the MNT-1 melanoma cell line model was investigated. Furthermore, related mechanisms were identified using colorimetric tyrosinase assay and the Western blot technique. The ACCE was composed of L-anserine and L-carnosine as two major constituents. In a dose-dependent manner, ACCE, L-anserine, and L-carnosine manifested significant antioxidation potential and significant reduction of melanin production. Activation of the extracellular signal-regulated kinase (ERK) signaling pathway and inhibition of tyrosinase activity of ACCE were demonstrated as the mechanisms of the anti-melanogenesis effect. In conclusion, ACCE has been revealed as a potential cosmeceutical agent due to its antioxidation and anti-melanogenic activity in association with L-anserine and L-carnosine composition and biomolecular regulating ability. Therefore, further studies and development should be considered to support the utilization of anserine/carnosine-rich chicken extract in the cosmetic industry for economic value creation and sustainability.

A Novel Serum Glycobiomarker for Diagnosis and Prognosis of Cholangiocarcinoma Detected by Butea monosperma Agglutinin.

Plant lectins are widely used in medical glycosciences and glycotechnology. Many lectin-based techniques have been applied for the detection of disease-associated glycans and glycoconjugates. In this study, Butea monosperma agglutinin (BMA), a lectin purified from seeds of the medicinal plant Butea monosperma, was used for the detection of cholangiocarcinoma (CCA)-associated glycans. Expression of BMA-binding N-acetyl galactosamine/galactose (GalNAc/Gal)-associated glycan (BMAG) in CCA tissues was determined using BMA lectin histochemistry; the results showed that BMAG was undetectable in normal bile ducts and drastically increased in preneoplastic bile ducts and CCA. The study in hamsters showed that an increase of BMAG was associated with carcinogenesis of CCA. Using an in-house double BMA sandwich enzyme-linked lectin assay, BMAG was highly detected in the sera of CCA patients. The level of serum BMAG in CCA patients (N = 83) was significantly higher than non-CCA controls (N = 287) and it was applicable for diagnosis of CCA with 55.4% sensitivity, 81.9% specificity, and 76.0% accuracy. A high level of serum BMAG (≥82.5 AU/mL) was associated with unfavorable survival of CCA patients; this information suggested the potential of serum BMAG as a poor prognostic indicator of CCA. In summary, BMAG was aberrantly expressed in preneoplastic bile ducts and CCA, it was also highly detected in patient serum which potentially used as a marker for diagnosis and prognostic prediction of CCA.

Carbohydrate Antigen 50: Values for Diagnosis and Prognostic Prediction of Intrahepatic Cholangiocarcinoma.

Background and objectives: Cancer-associated carbohydrate antigen 50 (CA50) is a marker for detection of gastrointestinal cancers, especially of pancreatic and colon cancer. In this study, the power of CA50 as a diagnostic and prognostic marker was evaluated in intrahepatic cholangiocarcinoma (iCCA). Materials and Methods: Serum CA50 levels of iCCA patients and non-cholangiocarcinoma controls (non-CCA, including healthy persons and patients with benign biliary diseases and other gastrointestinal cancers) were measured using MAGLUMI®800 CLIA analyzer. Diagnostic and prognostic values of serum CA50 levels were evaluated. Results: CA50 levels in the sera of iCCA patients were significantly higher than those of non-CCA controls (p < 0.001, Mann-Whitney U test). Using cut-off value of 25 U/mL, CA50 provided 65.9% sensitivity, 87.3% specificity, and 80.1% accuracy for diagnosis of iCCA. Serum CA50 levels were increased and associated with the severity of bile duct pathology. In addition, a higher level of CA50 was associated with poor clinical outcome and shorter survival in iCCA patients. Multivariate survival analysis by Cox regression model revealed the potential of CA50 as an independent poor prognostic indicator for iCCA, regardless of the age, sex, histological types, or tumor stages. Conclusions: CA50 can be a diagnostic and poor prognostic marker candidate for iCCA.

A Barley Efflux Transporter Operates in a Na+-Dependent Manner, as Revealed by a Multidisciplinary Platform.

Plant growth and survival depend upon the activity of membrane transporters that control the movement and distribution of solutes into, around, and out of plants. Although many plant transporters are known, their intrinsic properties make them difficult to study. In barley (Hordeum vulgare), the root anion-permeable transporter Bot1 plays a key role in tolerance to high soil boron, facilitating the efflux of borate from cells. However, its three-dimensional structure is unavailable and the molecular basis of its permeation function is unknown. Using an integrative platform of computational, biophysical, and biochemical tools as well as molecular biology, electrophysiology, and bioinformatics, we provide insight into the origin of transport function of Bot1. An atomistic model, supported by atomic force microscopy measurements, reveals that the protein folds into 13 transmembrane-spanning and five cytoplasmic α-helices. We predict a trimeric assembly of Bot1 and the presence of a Na(+) ion binding site, located in the proximity of a pore that conducts anions. Patch-clamp electrophysiology of Bot1 detects Na(+)-dependent polyvalent anion transport in a Nernstian manner with channel-like characteristics. Using alanine scanning, molecular dynamics simulations, and transport measurements, we show that conductance by Bot1 is abolished by removal of the Na(+) ion binding site. Our data enhance the understanding of the permeation functions of Bot1.

Increase of MAL-II Binding Alpha2,3-Sialylated Glycan Is Associated with 5-FU Resistance and Short Survival of Cholangiocarcinoma Patients.

BACKGROUND AND OBJECTIVES: Sialylation plays important roles in tumor progression. Our present study aimed to demonstrate the alteration of sialylation and its role in cholangiocarcinoma (CCA). MATERIALS AND METHODS: The α2,3- and α2,6-sialylation in CCA tissue was analyzed by lectin-histochemistry using Maackia amurensis lectin-II (MAL-II) and Sambucus nigra agglutinin (SNA). CCA cell lines were treated with the pan-sialylation inhibitor 3Fax-peracetyl-Neu5Ac (3F-Sia) followed by proliferation and chemosensitivity assays. RESULTS: MAL-II binding α2,3-Sialylated Glycan (MAL-SG) and SNA binding α2,6-Sialylated Glycan (SNA-SG) were both elevated in CCA compared with hyperplastic/dysplastic (HP/DP) and normal bile ducts (NBD). The positive staining for MAL-SG or SNA-SG were found in 82% (61/74) of the CCA cases. Higher expression of MAL-SG in CCA was associated with shorter survival of the patients. The median survival of patients with high and low MAL-SG were 167 and 308 days, respectively, with overall survival of 233 days, suggesting the involvement of MAL-SG in CCA progression. MAL-SG expression of CCA cell lines was markedly decreased after treatment with 3F-Sia for 48 to 72 h. While proliferation of CCA cells were not affected by 3F-Sia treatment, their susceptibility to 5-fluorouracil (5-FU) was significantly enhanced. These results suggest that sialylation is involved in the development of 5-FU resistance and the sialylation inhibitor 3F-Sia can be used as a chemosensitizer for CCA. CONCLUSIONS: Sialylation is critically involved in the development of chemoresistance of CCA, and sialylation inhibitors may be used as a chemosensitizer in CCA treatment.

The wheat TabZIP2 transcription factor is activated by the nutrient starvation-responsive SnRK3/CIPK protein kinase.

KEY MESSAGE: The understanding of roles of bZIP factors in biological processes during plant development and under abiotic stresses requires the detailed mechanistic knowledge of behaviour of TFs. Basic leucine zipper (bZIP) transcription factors (TFs) play key roles in the regulation of grain development and plant responses to abiotic stresses. We investigated the role and molecular mechanisms of function of the TabZIP2 gene isolated from drought-stressed wheat plants. Molecular characterisation of TabZIP2 and derived protein included analyses of gene expression and its target promoter, and the influence of interacting partners on the target promoter activation. Two interacting partners of TabZIP2, the 14-3-3 protein, TaWIN1 and the bZIP transcription factor TaABI5L, were identified in a Y2H screen. We established that under elevated ABA levels the activity of TabZIP2 was negatively regulated by the TaWIN1 protein and positively regulated by the SnRK3/CIPK protein kinase WPK4, reported previously to be responsive to nutrient starvation. The physical interaction between the TaWIN1 and the WPK4 was detected. We also compared the influence of homo- and hetero-dimerisation of TabZIP2 and TaABI5L on DNA binding. TabZIP2 gene functional analyses were performed using drought-inducible overexpression of TabZIP2 in transgenic wheat. Transgenic plants grown under moderate drought during flowering, were smaller than control plants, and had fewer spikes and seeds per plant. However, a single seed weight was increased compared to single seed weights of control plants in three of four evaluated transgenic lines. The observed phenotypes of transgenic plants and the regulation of TabZIP2 activity by nutrient starvation-responsive WPK4, suggest that the TabZIP2 could be the part of a signalling pathway, which controls the rearrangement of carbohydrate and nutrient flows in plant organs in response to drought.

Overexpression of the class I homeodomain transcription factor TaHDZipI-5 increases drought and frost tolerance in transgenic wheat.

Characterization of the function of stress-related genes helps to understand the mechanisms of plant responses to environmental conditions. The findings of this work defined the role of the wheat TaHDZipI-5 gene, encoding a stress-responsive homeodomain-leucine zipper class I (HD-Zip I) transcription factor, during the development of plant tolerance to frost and drought. Strong induction of TaHDZipI-5 expression by low temperatures, and the elevated TaHDZipI-5 levels of expression in flowers and early developing grains in the absence of stress, suggests that TaHDZipI-5 is involved in the regulation of frost tolerance at flowering. The TaHDZipI-5 protein behaved as an activator in a yeast transactivation assay, and the TaHDZipI-5 activation domain was localized to its C-terminus. The TaHDZipI-5 protein homo- and hetero-dimerizes with related TaHDZipI-3, and differences between DNA interactions in both dimers were specified at 3D molecular levels. The constitutive overexpression of TaHDZipI-5 in bread wheat significantly enhanced frost and drought tolerance of transgenic wheat lines with the appearance of undesired phenotypic features, which included a reduced plant size and biomass, delayed flowering and a grain yield decrease. An attempt to improve the phenotype of transgenic wheat by the application of stress-inducible promoters with contrasting properties did not lead to the elimination of undesired phenotype, apparently due to strict spatial requirements for TaHDZipI-5 overexpression.

Overexpression of the TaSHN1 transcription factor in bread wheat leads to leaf surface modifications, improved drought tolerance, and no yield penalty under controlled growth conditions.

Transcription factors regulate multiple networks, mediating the responses of organisms to stresses, including drought. Here, we investigated the role of the wheat transcription factor TaSHN1 in crop growth and drought tolerance. TaSHN1, isolated from bread wheat, was characterized for molecular interactions and functionality. The overexpression of TaSHN1 in wheat was followed by the evaluation of T2 and T3 transgenic lines for drought tolerance, growth, and yield components. Leaf surface changes were analysed by light microscopy, SEM, TEM, and GC-MS/GC-FID. TaSHN1 behaves as a transcriptional activator in a yeast transactivation assay and binds stress-related DNA cis-elements, determinants of which were revealed using 3D molecular modelling. The overexpression of TaSHN1 in transgenic wheat did not result in a yield penalty under the controlled plant growth conditions of a glasshouse. Transgenic lines had significantly lower stomatal density and leaf water loss and exhibited improved recovery after severe drought, compared with control plants. The comparative analysis of cuticular waxes revealed an increased accumulation of alkanes in leaves of transgenic lines. Our data demonstrate that TaSHN1 may operate as a positive modulator of drought stress tolerance. Positive attributes could be mediated through an enhanced accumulation of alkanes and reduced stomatal density.

Basic leucine zipper (bZIP) transcription factors involved in abiotic stresses: A molecular model of a wheat bZIP factor and implications of its structure in function.

BACKGROUND: Basic leucine zipper (bZIP) genes encode transcription factors (TFs) that control important biochemical and physiological processes in plants and all other eukaryotic organisms. SCOPE OF REVIEW: Here we present (i) the homo-dimeric structural model of bZIP consisting of basic leucine zipper and DNA binding regions, in complex with the synthetic Abscisic Acid-Responsive Element (ABREsyn); (ii) discuss homo- and hetero-dimerisation patterns of bZIP TFs; (iii) summarise the current progress in understanding the molecular mechanisms of function of bZIP TFs, including features determining the specificity of their binding to DNA cis-elements, and (iv) review information on interaction partners of bZIPs during plant development and stress response, as well as on types and roles of post-translational modifications, and regulatory aspects of protein-degradation mediated turn-over. Finally, we (v) recapitulate on the recent advances regarding functional roles of bZIP factors in major agricultural crops, and discuss the potential significance of bZIP-based genetic engineering in improving crop yield and tolerance to abiotic stresses. MAJOR CONCLUSIONS: An accurate analysis and understanding of roles of plant bZIP TFs in different biological processes requires the knowledge of interacting partners, time and location of expression in plant organs, and the information on mechanisms of homo- and hetero-dimerisation of bZIP TFs. GENERAL SIGNIFICANCE: Studies on molecular mechanisms of plant bZIP TFs at the atomic levels will provide novel insights into the regulatory processes during plant development, and responses to abiotic and biotic stresses.

Wheat drought-responsive WXPL transcription factors regulate cuticle biosynthesis genes.

The cuticle forms a hydrophobic waxy layer that covers plant organs and provides protection from biotic and abiotic stresses. Transcription of genes responsible for cuticle formation is regulated by several types of transcription factors (TFs). Five orthologous to WAX PRODUCTION (WXP1 and WXP2) genes from Medicago truncatula were isolated from a cDNA library prepared from flag leaves and spikes of drought tolerant wheat (Triticum aestivum, breeding line RAC875) and designated TaWXP-like (TaWXPL) genes. Tissue-specific and drought-responsive expression of TaWXPL1D and TaWXPL2B was investigated by quantitative RT-PCR in two Australian wheat genotypes, RAC875 and Kukri, with contrasting glaucousness and drought tolerance. Rapid dehydration and/or slowly developing cyclic drought induced specific expression patterns of WXPL genes in flag leaves of the two cultivars RAC875 and Kukri. TaWXPL1D and TaWXPL2B proteins acted as transcriptional activators in yeast and in wheat cell cultures, and conserved sequences in their activation domains were localised at their C-termini. The involvement of wheat WXPL TFs in regulation of cuticle biosynthesis was confirmed by transient expression in wheat cells, using the promoters of wheat genes encoding two cuticle biosynthetic enzymes, the 3-ketoacyl-CoA-synthetase and the cytochrome P450 monooxygenase. Using the yeast 1-hybrid (Y1H) assay we also demonstrated the differential binding preferences of TaWXPL1D and TaWXPL2B towards three stress-related DNA cis-elements. Protein structural determinants underlying binding selectivity were revealed using comparative 3D molecular modelling of AP2 domains in complex with cis-elements. A scheme is proposed, which links the roles of WXPL and cuticle-related MYB TFs in regulation of genes responsible for the synthesis of cuticle components.

Active site cleft mutants of Os9BGlu31 transglucosidase modify acceptor substrate specificity and allow production of multiple kaempferol glycosides.

BACKGROUND: Rice Os9BGlu31 is a transglucosidase that can transfer glucose to phenolic acids, flavonoids, and phytohormones. Os9BGlu31 displays a broad specificity with phenolic 1-O-ß-D-glucose esters acting as better glucose donors than glucosides, whereas the free phenolic acids of these esters are also excellent acceptor substrates. METHODS: Based on homology modeling of this enzyme, we made single point mutations of residues surrounding the acceptor binding region of the Os9BGlu31 active site. Products of the wild type and mutant enzymes in transglycosylation of phenolic acceptors from 4-nitrophenyl ß-D-glucopyranoside donor were identified and measured by UPLC and negative ion electrospray ionization tandem mass spectrometry (LCMSMS). RESULTS: The most active variant produced was W243N, while I172T and L183Q mutations decreased the activity, and other mutations at W243 (A, D, M, N, F and Y) had variable effects, depending on the acceptor substrate. The Os9BGlu31 W243N mutant activity was higher than that of wild type on phenolic acids and kaempferol, a flavonol containing 4 hydroxyl groups, and the wild type Os9BGlu31 produced only a single product from each of these acceptors in significant amounts, while W243 variants produced multiple glucoconjugates. Fragmentation analysis provisionally identified the kaempferol transglycosylation products as kaempferol 3-O, 7-O, and 4'-O glucosides and 3,7-O, 4',7-O, and 3,4'-O bis-O-glucosides. The Os9BGlu31 W243 mutants were also better able to use kaempferol 3-O-glucoside as a donor substrate. GENERAL SIGNIFICANCE: The W243 residue was found to be critical to the substrate and product specificity of Os9BGlu31 transglucosidase and mutation of this residue allows production of a range of glucoconjugates.

Change of function of the wheat stress-responsive transcriptional repressor TaRAP2.1L by repressor motif modification.

Plants respond to abiotic stresses by changes in gene regulation, including stress-inducible expression of transcriptional activators and repressors. One of the best characterized families of drought-related transcription factors are dehydration-responsive element binding (DREB) proteins, known as C-repeat binding factors (CBF). The wheat DREB/CBF gene TaRAP2.1L was isolated from drought-affected tissues using a dehydration-responsive element (DRE) as bait in a yeast one-hybrid screen. TaRAP2.1L is induced by elevated abscisic acid, drought and cold. A C-terminal ethylene responsive factor-associated amphiphilic repression (EAR) motif, known to be responsible for active repression of target genes, was identified in the TaRAP2.1L protein. It was found that TaRAP2.1L has a unique selectivity of DNA-binding, which differs from that of DREB activators. This binding selectivity remains unchanged in a TaRAP2.1L variant with an inactivated EAR motif (TaRAP2.1Lmut). To study the role of the TaRAP2.1L repressor activity associated with the EAR motif in planta, transgenic wheat overexpressing native or mutated TaRAP2.1L was generated. Overexpression of TaRAP2.1L under constitutive and stress-inducible promoters in transgenic wheat and barley led to dwarfism and decreased frost tolerance. By contrast, constitutive overexpression of the TaRAP2.1Lmut gene had little or no negative influence on wheat development or grain yield. Transgenic lines with the TaRAP2.1Lmut transgene had an enhanced ability to survive frost and drought. The improved stress tolerance is attributed to up-regulation of several stress-related genes known to be downstream genes of DREB/CBF activators.

Identification and characterization of wheat drought-responsive MYB transcription factors involved in the regulation of cuticle biosynthesis.

A plant cuticle forms a hydrophobic layer covering plant organs, and plays an important role in plant development and protection from environmental stresses. We examined epicuticular structure, composition, and a MYB-based regulatory network in two Australian wheat cultivars, RAC875 and Kukri, with contrasting cuticle appearance (glaucousness) and drought tolerance. Metabolomics and microscopic analyses of epicuticular waxes revealed that the content of ß-diketones was the major compositional and structural difference between RAC875 and Kukri. The content of ß-diketones remained the same while those of alkanes and primary alcohols were increased by drought in both cultivars, suggesting that the interplay of all components rather than a single one defines the difference in drought tolerance between cultivars. Six wheat genes encoding MYB transcription factors (TFs) were cloned; four of them were regulated in flag leaves of both cultivars by rapid dehydration and/or slowly developing cyclic drought. The involvement of selected MYB TFs in the regulation of cuticle biosynthesis was confirmed by a transient expression assay in wheat cell culture, using the promoters of wheat genes encoding cuticle biosynthesis-related enzymes and the SHINE1 (SHN1) TF. Two functional MYB-responsive elements, specifically recognized by TaMYB74 but not by other MYB TFs, were localized in the TdSHN1 promoter. Protein structural determinants underlying the binding specificity of TaMYB74 for functional DNA cis-elements were defined, using 3D protein molecular modelling. A scheme, linking drought-induced expression of the investigated TFs with downstream genes that participate in the synthesis of cuticle components, is proposed.

Rice Os9BGlu31 is a transglucosidase with the capacity to equilibrate phenylpropanoid, flavonoid, and phytohormone glycoconjugates.

Glycosylation is an important mechanism of controlling the reactivities and bioactivities of plant secondary metabolites and phytohormones. Rice (Oryza sativa) Os9BGlu31 is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized. The free acids of these compounds also served as the best acceptors, suggesting that Os9BGlu31 may equilibrate the levels of phenolic acids and carboxylated phytohormones and their glucoconjugates. The Os9BGlu31 gene is most highly expressed in senescing flag leaf and developing seed and is induced in rice seedlings in response to drought stress and treatment with phytohormones, including abscisic acid, ethephon, methyljasmonate, 2,4-dichlorophenoxyacetic acid, and kinetin. Although site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu(169)), catalytic nucleophile residues (Glu(387)), and His(386), the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH1 ß-glucosidases that utilize a double displacement retaining mechanism.

Structural analysis and insights into the glycon specificity of the rice GH1 Os7BGlu26 ß-D-mannosidase.

Rice Os7BGlu26 is a GH1 family glycoside hydrolase with a threefold higher kcat/Km value for 4-nitrophenyl ß-D-mannoside (4NPMan) compared with 4-nitrophenyl ß-D-glucoside (4NPGlc). To investigate its selectivity for ß-D-mannoside and ß-D-glucoside substrates, the structures of apo Os7BGlu26 at a resolution of 2.20 Šand of Os7BGlu26 with mannose at a resolution of 2.45 Šwere elucidated from isomorphous crystals in space group P212121. The (ß/α)8-barrel structure is similar to other GH1 family structures, but with a narrower active-site cleft. The Os7BGlu26 structure with D-mannose corresponds to a product complex, with ß-D-mannose in the (1)S5 skew-boat conformation. Docking of the (1)S3, (1)S5, (2)SO and (3)S1 pyranose-ring conformations of 4NPMan and 4NPGlc substrates into the active site of Os7BGlu26 indicated that the lowest energies were in the (1)S5 and (1)S3 skew-boat conformations. Comparison of these docked conformers with other rice GH1 structures revealed differences in the residues interacting with the catalytic acid/base between enzymes with and without ß-D-mannosidase activity. The mutation of Tyr134 to Trp in Os7BGlu26 resulted in similar kcat/Km values for 4NPMan and 4NPGlc, while mutation of Tyr134 to Phe resulted in a 37-fold higher kcat/Km for 4NPMan than 4NPGlc. Mutation of Cys182 to Thr decreased both the activity and the selectivity for ß-D-mannoside. It was concluded that interactions with the catalytic acid/base play a significant role in glycon selection.

The evolutionary advantage of an aromatic clamp in plant family 3 glycoside exo-hydrolases.

In the barley ß-D-glucan glucohydrolase, a glycoside hydrolase family 3 (GH3) enzyme, the Trp286/Trp434 clamp ensures ß-D-glucosides binding, which is fundamental for substrate hydrolysis during plant growth and development. We employ mutagenesis, high-resolution X-ray crystallography, and multi-scale molecular modelling methods to examine the binding and conformational behaviour of isomeric ß-D-glucosides during substrate-product assisted processive catalysis that operates in GH3 hydrolases. Enzyme kinetics reveals that the W434H mutant retains broad specificity, while W434A behaves as a strict (1,3)-ß-D-glucosidase. Investigations of reactant movements on the nanoscale reveal that processivity is sensitive to mutation-specific alterations of the tryptophan clamp. While wild-type and W434H utilise a lateral cavity for glucose displacement and sliding of (1,3)-linked hydrolytic products through the catalytic site without dissociation, consistent with their high hydrolytic rates, W434A does not adopt processive catalysis. Phylogenomic analyses of GH3 hydrolases disclose the evolutionary advantage of the tryptophan clamp that confers broad specificity, high catalytic efficiency, and processivity.

High-level expression of barley beta-D-glucan exohydrolase HvExoI from a codon-optimized cDNA in Pichia pastoris.

The native beta-d-glucan exohydrolase isoenzyme ExoI from barley seedlings, designated HvExoI, was the first GH3 glycoside hydrolase, for which a crystal structure was determined. A precise understanding of relationships between structure and function in this enzyme has been gained by structural and enzymatic studies. To allow testing of hypotheses gained from these studies, an efficient system for expression of HvExoI in Pichia pastoris was developed using a codon-optimized cDNA. Protein expression at a temperature of 20 degrees C yielded a recombinant enzyme, designated rHvExoI, which had molecular masses of 70-110 kDa due to heavy glycosylation at Asn221, Asn498 and Asn600, the three sites of N-glycosylation in native HvExoI. Most of the N-linked carbohydrate could be removed from rHvExoI, resulting in N-deglycosylated rHvExoI with a substantially decreased molecular mass of 67 kDa. rHvExoI was able to hydrolyse barley (1,3;1,4)-beta-D-glucan, laminarin and lichenans. The catalytic efficiency value k(cat)/K(M) of rHvExoI with barley (1,3;1,4)-beta-D-glucan was similar to that reported for native HvExoI. Further, laminaribiose, cellobiose and gentiobiose were formed through transglycosylation reactions with 4-nitrophenyl beta-D-glucoside and barley (1,3;1,4)-beta-D-glucan. Overall, the biochemical properties of rHvExoI were similar to those reported for native HvExoI, although differences were seen in thermostabilities and hydrolytic rates of certain beta-linked glucosides.