<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

Enzymatic treatment of XylT reaction products generated using Xyl₄-AA as acceptor.

<p>Microsomes were incubated with UDP-Xyl and the Xyl₄-AA acceptor, and the reaction products were then digested with endo-β-(1,4)-xylanase (B) and β-xylosidase (C). The reaction products with (B, C) and without (A) enzymatic digestion were analyzed by RP-HPLC.</p

Cell wall neutral polysaccharide components (mol percent) in fresh and stored asparagus spears.

<p>Cell wall neutral polysaccharide components (mol percent) in fresh and stored asparagus spears.</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

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

Comparison of xylan XylT activities in microsomes from <i>Arabidopsis</i>, <i>Asparagus</i> and barley (<i>Hordeum vulgare</i>).

<p>Microsomes from <i>Arabidopsis</i> stem, <i>Asparagus</i> spear and etiolated barley seedlings were isolated and the XylT activities were analyzed in the absence (-; black) and presence (+; grey) of the exogenous acceptor Xyl₆ according to the Materials and Methods.</p

Time course of processive transfer of Xyl residues onto the Xyl₄-AA acceptor.

<p><i>Asparagus</i> microsomes were incubated with UDP-Xyl and the Xyl₄-AA acceptor for 0.5 hr (B), 2 hr (C), 6 hr (D) and 10 hr (E). The reaction products were analyzed by RP-HPLC. (A) Standard chromatogram of Xyl₁-AA to Xyl₆-AA.</p

Biochemical properties of the xylan XylT activity in microsomes from garden asparagus spears.

<p>Microsomes extracted from the fresh basal <i>Asparagus</i> spears were incubated with UDP-[¹⁴C]-Xyl and Xyl₆ for 1 hr unless otherwise indicated. The XylT activity was measured by counting the radioactivity at the origin of the paper chromatogram and expressed as specific activity. All assays were repeated twice and the values were averaged. Effect of temperature (A), pH (B), reaction time (C), microsomal protein amount (D), amount of Xyl₆ (E) and UDP-Xyl concentration (F) on enzyme activity.</p

Xylan XylT activity measured using fluorescently tagged (AA) xylo-oligosaccharides of different lengths (Xyl₁-Xyl₆) as exogenous acceptors.

<p>The reaction was conducted by mixing <i>Asparagus</i> microsomes with cold UDP-Xyl and the fluorescent acceptors Xyl₁-AA (B), Xyl₂-AA (C), Xyl₃-AA (D), Xyl₄-AA (F), Xyl₅-AA (G) or Xyl₆-AA (H) and incubated at RT for 1 hr. The reaction products were separated by RP-HPLC and detected by a fluorescence detector. A chromatogram showing a separation of a Xyl₁-AA to Xyl₆-AA mixture of standards is shown in (A) and (E) for reference. The numbers on the plots indicates the DP of the xylo-AA oligosaccharides.</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