The biosynthesis and wall-binding of hemicelluloses in cellulose-deficient maize cells:An example of metabolic plasticity

Cell-suspension cultures（Zea mays L.,Black Mexican sweet corn） habituated to 2,6-dichlorobenzonitrile（DCB） survive with reduced cellulose owing to hemicellulose network modification. We aimed to de fine the hemicellulose metabolism modifications in DCB-habituated maize cells showing a mild reduction in cellulose at different stages in the culture cycle. Using pulse-chase radiolabeling, we fed habituated and non-habituated cultures with [3H]arabinose,and traced the distribution of 3H-pentose residues between xylans, xyloglucans and other polymers in several cellular compartments for 5 h. Habituated cells were slower taking up exogenous [3H]arabinose. Tritium was incorporated into polysaccharide-bound arabinose and xylose residues, but habituated cells diverted a higher proportion of their new [3H]xylose residues into（hetero） xylans at the expense of xyloglucan synthesis. During logarithmic growth, habituated cells showed slower vesicular traf ficking of polymers,especially xylans. Moreover, habituated cells showed a decrease in the strong wall-binding of all pentose-containing polysaccharides studied; correspondingly, especially in log phase cultures, habituation increased the proportion of 3H-hemicelluloses（[3H]xylans and [3H]xyloglucan） sloughed into the medium. These findings could be related to the cel walls’ cellulose-deficiency, and consequent reduction in binding sites for hemicelluloses; the data could also re fl ect the habituated cells’ reduced capacity to integrate arabinox ylans by extra-protoplasmic phenolic cross-linking, as well as xyloglucans, during wall assembly.

Hetero-trans-β-Glucanase Produces Cellulose-Xyloglucan Covalent Bonds in the Cell Walls of Structural Plant Tissues and Is Stimulated by Expansin

Current cell-wall models assume no covalent bonding between cellulose and hemicelluloses such as xyloglu-can or mixed-linkageβ-D-glucan(MLG).However,Equisetum hetero-trans-β-glucanase(HTG)grafts cellu-lose onto xyloglucan oligosaccharides(XGOs)-and,we now show,xyloglucan polysaccharide-in vitro,thus exhibiting CXE(cellulose:xyloglucan endotransglucosylase)activity.In addition,HTG also catalyzes MLG-to-XGO bonding(MXE activity).In this study,we explored the CXE action of HTG in native plant cell walls and tested whether expansin exposes cellulose to HTG by disrupting hydrogen bonds.To quantify and visu-alize CXE and MXE action,we assayed the sequential release of HTG products from cell walls pre-labeled with substrate mimics.We demonstrated covalent cellulose--xyloglucan bonding in plant cell walls and showed that CXE and MXE action was up to 15%and 60%of total transglucanase action,respectively,and peaked in aging,strengthening tissues:CXE in xylem and cells bordering intercellular canals and MXE in scleren-chyma.Recombinant bacterial expansin(EXLX1)strongly augmented CXE activity in vitro.CXE and MXE ac-tion in living Equisetum structural tissues potentially strengthens stems,while expansin might augment the HTG-catalyzed CXE reaction,thereby allowing efficient CXE action in muro.Our methods will enable surveys for comparable reactions throughout the plant kingdom.Furthermore,engineering similar hetero-polymer formation into angiosperm crop plants may improve certain agronomic traits such as lodging tolerance.

A general method for assaying homo-and hetero-transglycanase activities that act on plant cell-wall polysaccharides

Transglycanases（endotransglycosylases） cleave a polysaccharide（donor-substrate） in mid-chain, and then transfer a portion onto another poly-or oligosaccharide（acceptor-substrate）. Such enzymes contribute to plant cellwall assembly and/or re-structuring. We sought a general method for revealing novel homo- and hetero-transglycanases, applicable to diverse polysaccharides and oligosaccharides, separating transglycanase-generated3 Hpolysaccharides from unreacted3H-oligosaccharides—the former immobilized（on filter-paper, silica-gel or glassfiber）,the latter eluted. On filter-paper, certain polysaccharides [e.g.（1!3, 1!4）-b-D-glucans] remained satisfactorily adsorbed when water-washed; others（e.g. pectins） were partially lost. Many oligosaccharides（e.g. arabinan-, galactan-, xyloglucan-based） were successfully eluted in appropriate solvents, but others（e.g. [3H]xylohexaitol, [3H]mannohexaitol[3H]cellohexaitol） remained immobile. On silica-gel, all3 Holigosaccharides left an immobile ‘ghost’ spot（contaminating any3H-polysaccharides）, which was diminished but not prevented by additives e.g. sucrose or Triton X-100. The best stratum was glassfiber（GF）, onto which the reactionmixture was dried then washed in 75% ethanol. Washing led to minimal loss or lateral migration of3H-polysaccharides if conducted by slow percolation of acidified ethanol. The effectiveness of GF-blotting was well demonstrated for Chara vulgaris transb-mannanase. In conclusion, our novel GF-blotting technique ef ficiently frees transglycanase-generated3H-polysaccharides from unreacted3H-oligosaccharides,enabling high-throughput screening of multiple postulated transglycanase activities utilising chemically diverse donorand acceptor-substrates.

Feruloylated Arabinoxylans Are Oxidatively Cross- Linked by Extracellular Maize Peroxidase but Not by Horseradish Peroxidase

Covalent cross-linking of soluble extracellular arabinoxylans in living maize cultures, which models the cross- linking of wall-bound arabinoxylans, is due to oxidation of feruloyl esters to oligoferuloyl esters and ethers. The oxidizing system responsible could be H2O2/peroxidase, O2/laccase, or reactive oxygen species acting non-enzymically, To distinguish these possibilities, we studied arabinoxylan cross-linking in vivo and in vitro. In living cultures, exogenous, soluble, extracellular, feruloylated [pentosyl-3H]arabinoxylans underwent cross-linking, beginning abruptly 8 d after sub-culture. Crosslinking was suppressed by iodide, an H2O2 scavenger, indicating dependence on endogenous H2O2. However, exogenous H2O2 did not cause precocious cross-linking, despite the constant presence of endogenous peroxidases, suggesting that younger cultures contained natural cross-linking inhibitors. Dialysed culture-filtrates cross-linked [^3H]arabinoxylans in vitro only if H2O2 was also added, indicating a peroxidase requirement. This cross-linking was highly ionic-strength-dependent. The peroxidases responsible were heat-labile, although relatively heat-stable peroxidases （assayed on o-dianisidine） were also present. Surprisingly, added horseradish peroxidase, even after heat-denaturation, blocked the arabinoxylancross-linking action of maize peroxidases, suggesting that the horseradish protein was a competing substrate for [^3H]arabinoxylan coupling. In conclusion, we show for the first time that cross-linking of extracellular arabinoxylan in living maize cultures is an action of apoplastic peroxidases, some of whose unusual properties we report.

Changes in Cinnamic Acid Derivatives Associated with the Habituation of Maize Cells to Dichlobenil

The habituation of cell cultures to cellulose biosynthesis inhibitors such as dichlobenil （DCB） represents a valu- able tool to improve our knowledge of the mechanisms involved in plant cell wall structural plasticity. Maize cell lines habituated to lethal concentrations of DCB were able to grow through the acquisition of a modified cell wall in which cellulose was partially replaced by a more extensive network of arabinoxylans. The aim of this work was to investigate the phenolic metabolism of non-habituated and DCB-habituated maize cell cultures. Maize cell cultures were fed [14C]cinnamate and the fate of the radioactivity in different intra-protoplasmic and wall-localized fractions throughout the culture cycle was analyzed by autoradiography and scintillation counting. Non-habituated and habituated cultures did not markedly differ in their ability to uptake exogenous [14C]cinnamic acid. However, interesting differences were found in the radiolabeling of low- and high-Mr metabolites. Habituated cultures displayed a higher number and amount of radiola-beled low-Mr compounds, which could act as reserves later used for polysaccharide feruloylation. DCB-habituated cultures were highly enriched in esterified [14C]dehydrodiferulates and larger coupling products. In conclusion, an extensive and early cross-linking of hydroxycinnamates was observed in DCB-habituated cultures, probably strengthening their cellulose-deficient walls.

Phenolic metabolism and molecular mass distribution of polysaccharides in cellulose-deficient maize cells

As a consequence of the habituation to low levels of dichlobenil （DCB）, cultured maize cells presented an altered hemicellulose cell fate with a lower proportion of strongly wall-bound hemicelluloses and an increase in soluble extracellular polymers released into the culture medium. The aim of this study was to investigate the relative molecular mass distributions of polysaccharides as well as phenolic metabolism in cells habituated to low levels of DCB （1.5 μM）. Generally, cell wall bound hemicelluloses and sloughed polymers from habituated cells were more homogeneously sized and had a lower weight-average relative molecular mass. In addition, polysaccharides underwent massive cross-linking after being secreted into the cell wall, but this cross-linking was less pronounced in habituated cells than in non-habituated ones. However, when relativized, ferulic acid and p-coumaric acid contents were higher in this habituated cell line. Feasibly, cells habituated to low levels of DCB synthesized molecules with a lower weight-average relative molecular mass, although cross-linked, as a part of their strategy to compensate for the lack of cellulose.