Characterisation of soluble and insoluble cell wall fractions from rye, wheat and hull-less barley endosperm flours

Within cereal endosperm flours, arabinoxylan and β-glucan molecules exist in either a soluble or an insoluble form. From a nutritional functionality viewpoint, soluble and insoluble forms offer different potential health advantages, so it is important to define both the features controlling solubilisation and the properties of each of the soluble and insoluble fractions. Factors known to affect the stability of arabinoxylan (AX) and β-glucan (BG) solutions include AX branching extent and type, and the ratio of cellotriose to cellotetraose units (DP3/DP4) in BG. Through studying the solubilisation of AX and BG from wheat, rye, and hull less barley endosperm under conditions that avoid the use of alkali or ethanol during the solubilisation process, we report (a) similar A/X ratios and fine structures for extracted soluble arabinoxylan and the corresponding insoluble AX within the cell walls for rye and wheat endosperm flours, (b) comparable DP3/DP4 ratios for soluble β-glucan, flour and insoluble β-glucan within the endosperm cell wall of hull less barley, and (c) evidence for enrichment of β-glucan at the exterior of residual insoluble cell walls. Therefore, the factors determining solubilisation of AX and BG from endosperm cell walls are different to those that determine the stability of aqueous solutions of the same polymers, and β-glucan may show limited solubilisation by being trapped within restraining cross-linked arabinoxylans in the cell wall

Regulation of Meristem Morphogenesis by Cell Wall Synthases in Arabidopsis.

The cell walls of the shoot apical meristem (SAM), containing the stem cell niche that gives rise to the above-ground tissues, are crucially involved in regulating differentiation. It is currently unknown how these walls are built and refined or their role, if any, in influencing meristem developmental dynamics. We have combined polysaccharide linkage analysis, immuno-labeling, and transcriptome profiling of the SAM to provide a spatiotemporal plan of the walls of this dynamic structure. We find that meristematic cells express only a core subset of 152 genes encoding cell wall glycosyltransferases (GTs). Systemic localization of all these GT mRNAs by in situ hybridization reveals members with either enrichment in or specificity to apical subdomains such as emerging flower primordia, and a large class with high expression in dividing cells. The highly localized and coordinated expression of GTs in the SAM suggests distinct wall properties of meristematic cells and specific differences between newly forming walls and their mature descendants. Functional analysis demonstrates that a subset of CSLD genes is essential for proper meristem maintenance, confirming the key role of walls in developmental pathways.V.C. is in receipt of a Thailand Research Fund (TRF) grant for New Researcher (Grant Number TRG5880067), and a Research Supplement grant from Faculty of Science, Mahidol University. CB, MSD and AB acknowledge the support of the ARC Centre of Excellence in Plant Cell Walls, Australia (Grant Number CE110001007). EMM acknowledges support from the Gatsby Charitable Trust through Fellowships GAT3272/C and GAT3273-PR1, the Howard Hughes Medical Institute, the Gordon and Betty Moore Foundation (through Grant GBMF3406) and the US Department of Energy (through award DE-FG02-99ER13873). AP acknowledges support of the EU Marie-Curie FP7 COFUND People Programme through the award of an AgreenSkills grant no. 267196. RW acknowledges support from the Leverhulme Trust (Grant RPG-2015-285).This is the author accepted manuscript. The final version is available from Cell Press via http://dx.doi.org/10.1016/j.cub.2016.04.02

Prospecting for Energy-Rich Renewable Raw Materials: \u3cem\u3eAgave\u3c/em\u3e Leaf Case Study

Plant biomass from different species is heterogeneous, and this diversity in composition can be mined to identify materials of value to fuel and chemical industries. Agave produces high yields of energy-rich biomass, and the sugar-rich stem tissue has traditionally been used to make alcoholic beverages. Here, the compositions of Agave americana and Agave tequilana leaves are determined, particularly in the context of bioethanol production. Agave leaf cell wall polysaccharide content was characterized by linkage analysis, non-cellulosic polysaccharides such as pectins were observed by immuno-microscopy, and leaf juice composition was determined by liquid chromatography. Agave leaves are fruit-like--rich in moisture, soluble sugars and pectin. The dry leaf fiber was composed of crystalline cellulose (47-50% w/w) and non-cellulosic polysaccharides (16-22% w/w), and whole leaves were low in lignin (9-13% w/w). Of the dry mass of whole Agave leaves, 85-95% consisted of soluble sugars, cellulose, non-cellulosic polysaccharides, lignin, acetate, protein and minerals. Juice pressed from the Agave leaves accounted for 69% of the fresh weight and was rich in glucose and fructose. Hydrolysis of the fructan oligosaccharides doubled the amount of fermentable fructose in A. tequilana leaf juice samples and the concentration of fermentable hexose sugars was 41-48 g/L. In agricultural production systems such as the tequila making, Agave leaves are discarded as waste. Theoretically, up to 4000 L/ha/yr of bioethanol could be produced from juice extracted from waste Agave leaves. Using standard Saccharomyces cerevisiae strains to ferment Agave juice, we observed ethanol yields that were 66% of the theoretical yields. These data indicate that Agave could rival currently used bioethanol feedstocks, particularly if the fermentation organisms and conditions were adapted to suit Agave leaf composition

In vitro grown pollen tubes of Nicotiana alata actively synthesise a fucosylated xyloglucan.

Nicotiana alata pollen tubes are a widely used model for studies of polarized tip growth and cell wall synthesis in plants. To better understand these processes, RNA-Seq and de novo assembly methods were used to produce a transcriptome of N. alata pollen grains. Notable in the reconstructed transcriptome were sequences encoding proteins that are involved in the synthesis and remodelling of xyloglucan, a cell wall polysaccharide previously not thought to be deposited in Nicotiana pollen tube walls. Expression of several xyloglucan-related genes in actively growing pollen tubes was confirmed and xyloglucan epitopes were detected in the wall with carbohydrate-specific antibodies: the major xyloglucan oligosaccharides found in N. alata pollen grains and tubes were fucosylated, an unusual structure for the Solanaceae, the family to which Nicotiana belongs. Finally, carbohydrate linkages consistent with xyloglucan were identified chemically in the walls of N. alata pollen grains and pollen tubes grown in culture. The presence of a fucosylated xyloglucan in Nicotiana pollen tube walls was thus confirmed. The consequences of this discovery to models of pollen tube growth dynamics and more generally to polarised tip-growing cells in plants are discussed

Role of UDP-Glucuronic Acid Decarboxylase in Xylan Biosynthesis in Arabidopsis

UDP-xylose (UDP-Xyl) is the Xyl donor used in the synthesis of major plant cell-wall polysaccharides such as xylan (as a backbone-chain monosaccharide) and xyloglucan (as a branching monosaccharide). The biosynthesis of UDP-Xyl from UDP-glucuronic acid (UDP-GlcA) is irreversibly catalyzed by UDPglucuronic acid decarboxylase (UXS). Until now, little has been known about the physiological roles of UXS in plants. Here, we report that AtUXS1, AtUXS2, and AtUXS4 are located in the Golgi apparatus whereas AtUXS3, AtUXS5, and AtUXS6 are located in the cytosol. Although all six single AtUXS T-DNA mutants and the uxs1 usx2 uxs4 triple mutant show no obvious phenotype, the uxs3 uxs5 uxs6 triple mutant has an irregular xylem phenotype. Monosaccharide analysis showed that Xyl levels decreased in uxs3 uxs5 uxs6 and linkage analysis confirmed that the xylan content in uxs3 xus5 uxs6 declined, indicating that UDP-Xyl from cytosol AtUXS participates in xylan synthesis. Gel-permeation chromatography showed that the molecular weight of non-cellulosic polysaccharides in the triple mutants, mainly composed of xylans, is lower than that in the wild type, suggesting an effect on the elongation of the xylan backbone. Upon saccharification treatment stems of the uxs3 uxs5 uxs6 triple mutants released monosaccharides with a higher efficiency than those of the wild type. Taken together, our results indicate that the cytosol UXS plays a more important role than the Golgi-localized UXS in xylan biosynthesis

<i>Asparagus</i> Spears as a Model to Study Heteroxylan Biosynthesis during Secondary Wall Development

<div><p>Garden asparagus (<i>Asparagus officinalis</i> L.) is a commercially important crop species utilized for its excellent source of vitamins, minerals and dietary fiber. However, after harvest the tissue hardens and its quality rapidly deteriorates because spear cell walls become rigidified due to lignification and substantial increases in heteroxylan content. This latter observation prompted us to investigate the <i>in vitro</i> xylan xylosyltransferase (XylT) activity in asparagus. The current model system for studying heteroxylan biosynthesis, <i>Arabidopsis</i>, whilst a powerful genetic system, displays relatively low xylan XylT activity in <i>in vitro</i> microsomal preparations compared with garden asparagus therefore hampering our ability to study the molecular mechanism(s) of heteroxylan assembly. Here, we analyzed physiological and biochemical changes of garden asparagus spears stored at 4 °C after harvest and detected a high level of xylan XylT activity that accounts for this increased heteroxylan. The xylan XylT catalytic activity is at least thirteen-fold higher than that reported for previously published species, including <i>Arabidopsis</i> and grasses. A biochemical assay was optimized and up to seven successive Xyl residues were incorporated to extend the xylotetraose (Xyl₄) acceptor backbone. To further elucidate the xylan biosynthesis mechanism, we used RNA-seq to generate an <i>Asparagus</i> reference transcriptome and identified five putative xylan biosynthetic genes (<i>AoIRX9</i>, <i>AoIRX9-L</i>, <i>AoIRX10</i>, <i>AoIRX14_A</i>, <i>AoIRX14_B</i>) with <i>AoIRX9</i> having an expression profile that is distinct from the other genes. We propose that <i>Asparagus</i> provides an ideal biochemical system to investigate the biochemical aspects of heteroxylan biosynthesis and also offers the additional benefit of being able to study the lignification process during plant stem maturation.</p></div

Physiological characteristics of fresh and stored <i>Asparagus</i> spears during low temperature (4°C) storage.

<p>(A) Changes in fresh weight, (B) visual appearance index, (C) texture (firmness) and (D) lignin content. The data are the average of fifteen <i>Asparagus</i> spears.</p

MALDI-TOF mass spectra of the xylan XylT reaction products catalyzed by <i>Asparagus</i> spear microsomes.

<p>Microsomes were incubated with UDP-Xyl and the Xyl₅-AA acceptor for 1 hr. Reaction products were purified by RP-HPLC and the fraction between 12 and 15 min collected and analyzed by MALDI-TOF MS. The ions corresponding to AA-labeled xylo-oligosaccharides (both in H⁺ and Na⁺ form) are labelled.</p

Xylan XylT activities of different <i>Asparagus</i> sections over the 4°C storage period.

<p>Microsomes from apical, middle and basal sections of <i>Asparagus</i> spears stored at 4°C for 0–16 days were isolated and the XylT activities were measured as described in the Materials and Methods. Data were average values±SE (n = 3).</p

XylT reaction products generated using Xyl₆ as the acceptor were fractionated on a Bio-Gel P2 column.

<p><i>Asparagus</i> microsomes were incubated with UDP-[¹⁴C]-Xyl and Xyl₆ acceptor. The reaction products were treated with Dowex resin to remove unreacted UDP-[¹⁴C]-Xyl and fractionated on Bio-Gel P2 column. V₀ and Vt, void and total volume, respectively. X₆ and X3, xylo-hexaoligosaccharide and xylo-trioligosaccharide standards.</p