Combined meta-genomics analyses unravel candidate genes for the grain dietary fiber content in bread wheat (Triticum aestivum L.).

Grain dietary fiber content in wheat not only affects its end use and technological properties including milling, baking and animal feed but is also of great importance for health benefits. In this study, integration of association genetics (seven detected loci on chromosomes 1B, 3A, 3D, 5B, 6B, 7A, 7B) and meta-QTL (three consensus QTL on chromosomes 1B, 3D and 6B) analyses allowed the identification of seven chromosomal regions underlying grain dietary fiber content in bread wheat. Based either on a diversity panel or on bi-parental populations, we clearly demonstrate that this trait is mainly driven by a major locus located on chromosome 1B associated with a log of p value >13 and a LOD score >8, respectively. In parallel, we identified 73 genes differentially expressed during the grain development and between genotypes with contrasting grain fiber contents. Integration of quantitative genetics and transcriptomic data allowed us to propose a short list of candidate genes that are conserved in the rice, sorghum and Brachypodium chromosome regions orthologous to the seven wheat grain fiber content QTL and that can be considered as major candidate genes for future improvement of the grain dietary fiber content in bread wheat breeding programs.

2-D DIGE reveals changes in wheat xylanase inhibitor protein families due to Fusarium graminearum DeltaTri5 infection and grain development.

Wheat contains three different classes of proteinaceous xylanase inhibitors (XIs), i.e. Triticum aestivum xylanase inhibitors (TAXIs) xylanase-inhibiting proteins (XIPs), and thaumatin-like xylanase inhibitors (TLXIs) which are believed to act as a defensive barrier against phytopathogenic attack. In the absence of relevant data in wheat kernels, we here examined the response of the different members of the XI protein population to infection with a DeltaTri5 mutant of Fusarium graminearum, the wild type of which is one of the most important wheat ear pathogens, in early developing wheat grain. Wheat ears were inoculated at anthesis, analyzed using 2-D DIGE and multivariate analysis at 5, 15, and 25 days post anthesis (DPA), and compared with control samples. Distinct abundance patterns could be distinguished for different XI forms in response to infection with F. graminearum DeltaTri5. Some (iso)forms were up-regulated, whereas others were down-regulated. This pathogen-specific regulation of proteins was mostly visible at five DPA and levelled off in the samples situated further from the inoculation point. Furthermore, it was shown that most identified TAXI- and XIP-type XI (iso)forms significantly increased in abundance from the milky (15 DPA) to the soft dough stages (25 DPA) on a per kernel basis, although the extent of increase differed greatly. Non-glycosylated XIP forms increased more strongly than their glycosylated counterparts.

Biochemical and structural characterization of TLXI, the Triticum aestivum L. thaumatin-like xylanase inhibitor.

Thaumatin-like xylanase inhibitors (TLXI) are recently discovered wheat proteins. They belong to the family of the thaumatin-like proteins and inhibit glycoside hydrolase family 11 endoxylanases commonly used in different cereal based (bio)technological processes. We here report on the biochemical characterisation of TLXI. Its inhibition activity is temperature- and pH-dependent and shows a maximum at approximately 40 degrees C and pH 5.0. The TLXI structure model, generated with the crystal structure of thaumatin as template, shows the occurrence of five disulfide bridges and three beta-sheets. Much as in the structures of other short-chain thaumatin-like proteins, no alpha-helix is present. The circular dichroism spectrum of TLXI confirms the absence of alpha-helices and the presence of antiparallel beta-sheets. All ten cysteine residues in TLXI are involved in disulfide bridges. TLXI is stable for at least 120 min between pH 1-12 and for at least 2 hours at 100 degrees C, making it much more stable than the other two xylanase inhibitors from wheat, i.e. Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibitor protein (XIP). This high stability can probably be ascribed to the high number of disulfide bridges, much as seen for other thaumatin-like proteins.

His22 of TLXI plays a critical role in the inhibition of glycoside hydrolase family 11 xylanases.

Recently, a novel wheat thaumatin-like protein, TLXI, which inhibits microbial glycoside hydrolase family (GH) 11 xylanases has been identified. It is the first xylanase inhibitor that exerts its inhibition in a non-competitive way. In the present study we gained insight into the interaction between TLXI and xylanases via combined molecular modeling and mutagenic approaches. More specifically, site-specific mutation of His22, situated on a loop which distinguishes TLXI from other, non-inhibiting, thaumatin-like proteins, and subsequent expression of the mutant in Pichia pastoris resulted in a protein lacking inhibition capacity. The mutant protein was unable to form a complex with GH11 xylanases. Based on these findings, the interaction of TLXI with GH11 xylanases is discussed.

Variability of polymorphic families of three types of xylanase inhibitors in the wheat grain proteome.

Cereals contain proteinaceous inhibitors of endo-beta-1,4-xylanases (E.C.3.2.1.8, xylanases). Since these xylanase inhibitors (XIs) are only active against xylanases of microbial origin and do not interact with plant endogenous xylanases, they are believed to act as a defensive barrier against phytopathogenic attack. So far, three types of XIs have been identified, i.e. Triticum aestivum XI (TAXI), xylanase inhibiting protein (XIP), and thaumatin-like XI (TLXI) proteins. In this study the variation in XI forms present in wheat grain was elucidated using high-resolution 2-DE in combination with LC-ESI-MS/MS and biochemical techniques. Reproducible 2-DE fingerprints of TAXI-, XIP-, and TLXI-type XIs, selectively purified from whole meal of three European wheat cultivars using cation exchange chromatography followed by affinity chromatography, were obtained using a pH-gradient of 6 to 11 and a molecular mass range of 10 to 60 kDa. Large polymorphic XI families, not known to date, which exhibit different pI- and/or molecular mass values, were visualised by colloidal CBB staining. Identification of distinct genetic variants by MS/MS-analysis provides a partial explanation for the observed XI heterogeneity. Besides genetic diversity, PTMs, such as glycosylation, account for the additional complexity of the 2-DE patterns.

Contribution of wheat endogenous and wheat kernel associated microbial endoxylanases to changes in the arabinoxylan population during breadmaking.

Wheat kernel associated endoxylanases consist of a majority of microbial endoxylanases and a minority of endogenous endoxylanases. At least part of these enzymes can be expected to end up in wheat flour upon milling. In this study, the contribution of both types of these endoxylanases to changes in the arabinoxylan (AX) population during wheat flour breadmaking was assessed. To this end, wheat flour produced from two wheat varieties with different endoxylanase activity levels, both before and after sodium hypochlorite surface treatment of the wheat kernels, was used in a straight dough breadmaking procedure. Monitoring of the AX population during the breadmaking process showed that changes in AX are to a large extent caused by endogenous endoxylanases, whereas the contribution of microbial endoxylanases to these changes was generally very low. The latter points to a limited contamination of wheat flour with microbial enzymes during milling or to an extensive inactivation of these wheat flour associated microbial endoxylanases by endoxylanase inhibitors, present in wheat flour. When all wheat kernel associated microbial endoxylanases were first washed from the kernels and then added to the bread recipe, they drastically affected the AX population, suggesting that they can have a large impact on whole meal breadmaking.

Xylanase inhibitors bind to nonstarch polysaccharides.

This study is an in-depth investigation of the interaction between polysaccharides and the proteinaceous xylanase inhibitors, Triticum aestivum xylanase inhibitor (TAXI), xylanase inhibitor protein (XIP), and thaumatin-like xylanase inhibitor (TLXI). The binding affinities of all three known types of xylanase inhibitors from wheat are studied by measuring the residual xylanase inhibition activity after incubation of the inhibitors in the presence of different polysaccharides, such as beta-glucans and (arabino)xylans. The binding affinities of all three xylanase inhibitors for (arabino)xylans increased with a decreasing arabinose/xylose ratio (A/X ratio). This phenomenon was observed both with water-extractable and water-unextractable (arabino)xylans. The inhibitors also interacted with different soluble and insoluble beta-glucans. None of the inhibitors tested had the ability to hydrolyze the polysaccharides investigated. The present findings contribute to the unraveling of the function of xylanase inhibitors in nature and to the prediction of the effect of added xylanases in cereal-based biotechnological processes, such as bread making and gluten-starch separation.

TLXI, a novel type of xylanase inhibitor from wheat (Triticum aestivum) belonging to the thaumatin family.

Wheat (Triticum aestivum) contains a previously unknown type of xylanase (EC 3.2.1.8) inhibitor, which is described in the present paper for the first time. Based on its >60% similarity to TLPs (thaumatin-like proteins) and the fact that it contains the Prosite PS00316 thaumatin family signature, it is referred to as TLXI (thaumatin-like xylanase inhibitor). TLXI is a basic (pI> or =9.3 in isoelectric focusing) protein with a molecular mass of approx. 18-kDa (determined by SDS/PAGE) and it occurs in wheat with varying extents of glycosylation. The TLXI gene sequence encodes a 26-amino-acid signal sequence followed by a 151-amino-acid mature protein with a calculated molecular mass of 15.6-kDa and pI of 8.38. The mature TLXI protein was expressed successfully in Pichia pastoris, resulting in a 21-kDa (determined by SDS/PAGE) recombinant protein (rTLXI). Polyclonal antibodies raised against TLXI purified from wheat react with epitopes of rTLXI as well as with those of thaumatin, demonstrating high structural similarity between these three proteins. TLXI has a unique inhibition specificity. It is a non-competitive inhibitor of a number of glycoside hydrolase family 11 xylanases, but it is inactive towards glycoside hydrolase family 10 xylanases. Progress curves show that TLXI is a slow tight-binding inhibitor, with a K(i) of approx. 60-nM. Except for zeamatin, an alpha-amylase/trypsin inhibitor from maize (Zea mays), no other enzyme inhibitor is currently known among the TLPs. TLXI thus represents a novel type of inhibitor within this group of proteins.

Impact of parboiling conditions on Maillard precursors and indicators in long-grain rice cultivars.

The effect of steaming conditions (mild, intermediate and severe) during parboiling of five different long-grain rice cultivars (brown rice cultivars Puntal, Cocodrie, XL8 and Jacinto, and a red rice) on rice colour, and Maillard precursors and indicators was investigated. Rice colour increased with severity of parboiling conditions. Redness increased more than yellowness when parboiling brown rice. Parboiling turned red rice black. It changed the levels of glucose, fructose, sucrose, and maltose. Losses of the non-reducing sugar, sucrose were caused by both leaching into the soaking water and enzymic conversion, rather than by thermal degradation during steaming. Concentrations of the reducing sugars, glucose and fructose, in intermediately parboiled rice were higher than those of mildly parboiled rice. After severe parboiling, glucose levels were lower than those of intermediately parboiled rice, while fructose levels were higher. These changes were ascribed to the sum of losses in the Maillard reaction (MR), formations as a result of starch degradation and isomerisation of glucose into fructose. It was clear that the ε-amino group of protein-bound lysine was more affected by parboiling conditions and loss in MRs, than that of free lysine. Low values of the MR indicators furosine and free 5-hydroxymethyl-2-furaldehyde (HMF) in processed brown and red rices were related to mild parboiling, whereas high furosine and low free HMF levels were indicative of rices being subjected to intermediate processing conditions. High furosine and high free HMF contents corresponded to severe hydrothermal treatments. The strong correlation (r=0.89) between the free HMF levels and the increase in redness of parboiled brown rices suggested that Maillard browning was reflected more in the red than in the yellow colour.

Impact of wheat flour-associated endoxylanases on arabinoxylan in dough after mixing and resting.

The impact of varying levels of endoxylanase activity in wheat flour on arabinoxylan (AX) in mixed and rested dough was studied using eight industrially milled wheat flour fractions with varying endoxylanase activity levels. Analysis of the levels of reducing end xylose (RX) and solubilized AX (S-AX) formed during mixing and resting and their correlation with the endoxylanase activity in the flour milling fractions showed that solubilization of AX during the mixing phase is mainly due to mechanical forces, while solubilization of AX during resting is caused by endoxylanase activity. Moreover, solubilization of AX during the dough resting phase is more outspoken than that during the mixing phase. Besides endoxylanase activity, there were significant xylosidase and arabinofuranosidase activities during the dough resting phase. The results indicate that wheat flour-associated endoxylanases can alter part of the AX in dough, thereby changing their functionality in bread making and potentially affecting dough and end product properties.

Indirect enzyme-antibody sandwich enzyme-linked immunosorbent assay for quantification of TAXI and XIP type xylanase inhibitors in wheat and other cereals.

To quantify Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibiting protein (XIP) type proteins in cereals in general and wheat ( T. aestivum) in particular, a robust enzyme-linked immunosorbent assay (ELISA) using an uncommon enzyme-antibody sandwich format was developed. Bacillus subtilis glycoside hydrolase family (GH) 11 and Aspergillus oryzae GH 10 xylanases were selected for coating ELISA plate wells to capture TAXI and XIP, respectively, prior to probing with antibodies. The detection threshold of the developed ELISA was much lower than that of the currently used xylanase inhibitor assay and the recently described Western blot approach. Because of its broad dynamic range (TAXI, 30-600 ng/mL, and XIP, 3-60 ng/mL), one proper standard extract dilution can be used for analyzing different wheat varieties, whereas for the currently used colorimetric assay, often different dilutions need to be analyzed. The TAXI ELISA for wheat was successfully adapted for barley ( Hordeum vulgare) and could also be used for other cereals.

Insight into the distribution of arabinoxylans, endoxylanases, and endoxylanase inhibitors in industrial wheat roller mill streams.

To gain insight into the distribution of arabinoxylans (AX), endoxylanases, and endoxylanase inhibitors in industrial wheat roller milling, all streams, that is, 54 flour fractions, 4 bran fractions, and the germ, were analyzed for ash, starch, and protein contents, alpha-amylase activity levels, total (TOT-AX) and water-extractable arabinoxylan (WE-AX) contents, endoxylanase activity levels, and endoxylanase inhibitor (TAXI and XIP) contents. In general, bran fractions were significantly richer in TOT-AX and WE-AX contents, endoxylanase activity levels, and endoxylanase inhibitor contents than germ and, even more so, than flour fractions. In the 54 different flour fractions, minimal and maximal values for TOT-AX and WE-AX contents differed by ca. 2-fold, whereas they differed by ca. 15-fold for endoxylanase activity levels. The latter were positively correlated with ash and negatively correlated with starch content, suggesting that the endoxylanase activity in flour is strongly influenced by the level of bran contamination. TAXI contents in the flour fractions varied ca. 4-fold and were strongly correlated with bran-related parameters such as ash content and enzyme activity levels, whereas XIP contents varied ca. 3-fold and were not correlated with any of the parameters measured in this study. The results can be valuable in blending and optimizing wheat flour fractions to obtain flours with specific technological and nutritional benefits.

Wheat-kernel-associated endoxylanases consist of a majority of microbial and a minority of wheat endogenous endoxylanases.

The endoxylanases associated with wheat kernels consist of wheat endogenous endoxylanases on one hand and kernel-associated microbial endoxylanases on the other hand. Assessment of their presence, based on analysis of their enzymic activity, can be expected to be hampered by the presence in wheat of high levels of endogenous endoxylanase inhibitors, which are able to inhibit the wheat-kernel-associated microbial endoxylanases. On the basis of preliminary experiments aimed at clarifying the distribution of the wheat-associated endoxylanases, a method to estimate total endoxylanase activities in wheat kernels was developed. Extensive washing of wheat kernels with universal buffer of pH 8.0 provided near-quantitative separation of the microbial endoxylanases located on the surface of wheat kernels from the endogenous endoxylanases and endoxylanase inhibitors located in such kernels. The microbial or endogenous nature of the endoxylanases was confirmed by making use of the inhibition specificity of endoxylanase inhibitors. Determination of the endoxylanase activity in the washing liquid, corresponding to the microbial endoxylanase population, and the washed kernels, corresponding to the endogenous endoxylanase population, allowed estimation of the total endoxylanase activities associated with the wheat kernel. Results showed that microbial endoxylanases can account for over 90% of the total wheat-associated endoxylanase activity and that the latter can be at least 5 times higher than the apparent endoxylanase activity.

Occurrence of proteinaceous endoxylanase inhibitors in cereals.

Cereals contain proteinaceous inhibitors of endoxylanases, which affect the efficiency and functionality of these enzymes in cereal processing. This review relates their first discovery in wheat and the subsequent purification of two distinct classes of endoxylanase inhibitors, namely Triticum aestivum xylanase inhibitor (TAXI)-type and xylanase inhibitor protein (XIP)-type inhibitors in cereals. Both inhibitor classes occur in monocots as multi-isoform families. The reported data provide an overview of the relative quantitative and qualitative variation of these inhibitors in cereals. Wheat and rye are particularly rich in TAXI-type and XIP-type inhibitors with the latter inhibitors being more abundant. Lower inhibitor levels are present in durum wheat and barley, while maize contains solely XIP-type inhibitors. No inhibitors have been isolated from rice, oats and buckwheat.

A family 11 xylanase from Penicillium funiculosum is strongly inhibited by three wheat xylanase inhibitors.

Steady-state kinetic approaches were used to investigate the binding of a novel Penicillium funiculosum xylanase, XYNC, with three known xylanase inhibitor proteins from wheat (Triticum aestivum). The xylanase gene (xynC) was cloned from a P. funiculosum genomic library and the deduced amino acid sequence of XYNC exhibited high sequence similarity with fungal family 11 xylanases. xynC was overexpressed in P. funiculosum and the product (XYNC: M(r)=23.6 kDa; pI=3.7) purified and shown to efficiently degrade birchwood xylan [K(m)=0.47% w/v, Vmax=2540 micromol xylose min(-1) (mg protein)(-1) at pH 5.5 and 30 degrees C] and soluble wheat arabinoxylans [K(m)=1.45% w/v, Vmax=7190 micromol xylose min(-1) mg protein)(-1) at pH 5.5 and 30 degrees C]. The xylanase activity of XYNC was inhibited strongly by three xylanase inhibitor proteins from wheat; XIP-I, TAXI I and TAXI II. The inhibition for each was competitive, with very tight binding (K(i)=3.4, 16 and 17 nM, respectively) equivalent to free energy changes (deltaG degrees ) of -49, -45 and -45 kJ mol(-1). This is the first report describing a xylanase that is inhibited by all three wheat xylanase inhibitor proteins described to date.

Properties of TAXI-type endoxylanase inhibitors.

Two types of proteinaceous endoxylanase inhibitors occur in different cereals, i.e. the TAXI [Triticum aestivum endoxylanase inhibitor]-type and XIP [endoxylanase inhibiting protein]-type inhibitors. The present paper focuses on the TAXI-type proteins and deals with their structural characteristics and the identification, characterisation and heterologous expression of a TAXI gene from wheat. In addition, to shed light on the mechanism by which TAXI-type endoxylanase inhibitors work, the enzyme specificity, the optimal conditions for maximal inhibition activity, the molar complexation ratio and the inhibition kinetics of the inhibitors are explained and the effect of mutations of an endoxylanase on the inhibition by TAXIs is discussed.

Potential physiological role of plant glycosidase inhibitors.

Carbohydrate-active enzymes including glycosidases, transglycosidases, glycosyltransferases, polysaccharide lyases and carbohydrate esterases are responsible for the enzymatic processing of carbohydrates in plants. A number of carbohydrate-active enzymes are produced by microbial pathogens and insects responsible of severe crop losses. Plants have evolved proteinaceous inhibitors to modulate the activity of several of these enzymes. The continuing discovery of new inhibitors indicates that this research area is still unexplored and may lead to new exciting developments. To date, the role of the inhibitors is not completely understood. Here we review recent results obtained on the best characterised inhibitors, pointing to their possible biological role in vivo. Results recently obtained with plant transformation technology indicate that this class of inhibitors has potential biotechnological applications.

His374 of wheat endoxylanase inhibitor TAXI-I stabilizes complex formation with glycoside hydrolase family 11 endoxylanases.

Wheat endoxylanase inhibitor TAXI-I inhibits microbial glycoside hydrolase family 11 endoxylanases. Crystallographic data of an Aspergillus niger endoxylanase-TAXI-I complex showed His374 of TAXI-I to be a key residue in endoxylanase inhibition. Its role in enzyme-inhibitor interaction was further investigated by site-directed mutagenesis of His374 into alanine, glutamine or lysine. Binding kinetics and affinities of the molecular interactions between A. niger, Bacillus subtilis, Trichoderma longibrachiatumendoxylanases and wild-type TAXI-I and TAXI-I His374 mutants were determined by surface plasmon resonance analysis. Enzyme-inhibitor binding was in accordance with a simple 1 : 1 binding model. Association and dissociation rate constants of wild-type TAXI-I towards the endoxylanases were in the range between 1.96 and 36.1 x 10(4)m(-1) x s(-1) and 0.72-3.60 x 10(-4) x s(-1), respectively, resulting in equilibrium dissociation constants in the low nanomolar range. Mutation of TAXI-I His374 to a variable degree reduced the inhibition capacity of the inhibitor mainly due to higher complex dissociation rate constants (three- to 80-fold increase). The association rate constants were affected to a smaller extent (up to eightfold decrease). Substitution of TAXI-I His374 therefore strongly affects the affinity of the inhibitor for the enzymes. In addition, the results show that His374 plays a critical role in the stabilization of the endoxylanase-TAXI-I complex rather than in the docking of inhibitor onto enzyme.

Evidence for the involvement of arabinoxylan and xylanases in refrigerated dough syruping.

The relationship between syruping in refrigerated doughs upon prolonged storage and different aspects of arabinoxylan (AX) hydrolysis was investigated using Triticum aestivum xylanase inhibitor (TAXI) and different xylanases in the dough formula. Dough characteristics were evaluated with strong emphasis on the AX population and its fate as a function of storage time. Selective reduction of part of the flour endogenous xylanase activity in dough by added TAXI reduced dough syruping after 12 and 20 days of storage by 50%, providing straightforward evidence for the involvement of xylanases and, thus, AX in the syruping phenomenon. Addition of xylanases with different inhibitor sensitivities [an inhibition-sensitive Bacillus subtilis xylanase (XBS(i)) as well as a noninhibited mutant (XBS(ni)) thereof] to dough confirmed the importance of xylanases in dough syruping, on one hand, and the power of wheat flour TAXI to constitute a significant barrier against xylanase-mediated dough syruping, on the other hand. Use of xylanases with different substrate selectivities [an Aspergillus aculeatusxylanase (XAA) versus XBS(ni)] showed degradation of water-extractable AX (WE-AX) and solubilized AX to low molecular weight molecules rather than the conversion of water-unextractable AX (WU-AX) to high molecular weight water extractable components to be the main factor influencing dough syruping.