Rudy Vergauwen

Fructan 1-Exohydrolases. β-(2,1)-Trimmers during Graminan Biosynthesis in Stems of Wheat? Purification, Characterization, Mass Mapping, and Cloning of Two Fructan 1-Exohydrolase Isoforms

Graminan-type fructans are temporarily stored in wheat (Triticum aestivum) stems. Two phases can be distinguished: a phase of fructan biosynthesis (green stems) followed by a breakdown phase (stems turning yellow). So far, no plant fructan exohydrolase enzymes have been cloned from a monocotyledonous species. Here, we report on the cloning, purification, and characterization of two fructan 1-exohydrolase cDNAs (1-FEH w1 andw2) from winter wheat stems. Similar to dicot plant 1-FEHs, they are derived from a special group within the cell wall-type invertases characterized by their low isoelectric points. The corresponding isoenzymes were purified to electrophoretic homogeneity, and their mass spectra were determined by quadrupole-time-of-flight mass spectrometry. Characterization of the purified enzymes revealed that inulin-type fructans [β-(2,1)] are much better substrates than levan-type fructans [β-(2,6)]. Although both enzymes are highly identical (98% identity), they showed different substrate specificity toward branched wheat stem fructans. Although 1-FEH activities were found to be considerably higher during the fructan breakdown phase, it was possible to purify substantial amounts of 1-FEH w2 from young, fructan biosynthesizing wheat stems, suggesting that this isoenzyme might play a role as a β-(2,1)-trimmer throughout the period of active graminan biosynthesis. In this way, the species and developmental stage-specific complex fructan patterns found in monocots might be determined by the relative proportions and specificities of both fructan biosynthetic and breakdown enzymes.

Towards understanding vacuolar antioxidant mechanisms: a role for fructans?

Recent in vitro, in vivo, and theoretical experiments strongly suggest that sugar-(like) molecules counteract oxidative stress by acting as genuine reactive oxygen species (ROS) scavengers. A concept was proposed to include the vacuole as a part of the cellular antioxidant network. According to this view, sugars and sugar-like vacuolar compounds work in concert with vacuolar phenolic compounds and the ‘classic’ cytosolic antioxidant mechanisms. Among the biologically relevant ROS (H2O2, O2·–, and ·OH), hydroxyl radicals are the most reactive and dangerous species since there are no enzymatic systems known to neutralize them in any living beings. Therefore, it is important to study in more detail the radical reactions between ·OH and different biomolecules, including sugars. Here, Fenton reactions were used to compare the ·OH-scavenging capacities of a range of natural vacuolar compounds to establish relationships between antioxidant capacity and chemical structure and to unravel the mechanisms of ·OH–carbohydrate reactions. The in vitro work on the ·OH-scavenging capacity of sugars and phenolic compounds revealed a correlation between structure and ·OH-scavenging capacity. The number and position of the C=C type of linkages in phenolic compounds greatly influence antioxidant properties. Importantly, the splitting of disaccharides and oligosaccharides emerged as a predominant outcome of the ·OH–carbohydrate interaction. Moreover, non-enzymatic synthesis of new fructan oligosaccharides was found starting from 1-kestotriose. Based on these and previous findings, a working model is proposed describing the putative radical reactions involving fructans and secondary metabolites at the inner side of the tonoplast and in the vacuolar lumen.

Water stress drastically reduces root growth and inulin yield in Cichorium intybus (var. sativum) independently of photosynthesis

Root chicory (Cichorium intybus var. sativum) is a cash crop cultivated for inulin production in Western Europe. This plant can be exposed to severe water stress during the last 3 months of its 6-month growing period. The aim of this study was to quantify the effect of a progressive decline in water availability on plant growth, photosynthesis, and sugar metabolism and to determine its impact on inulin production. Water stress drastically decreased fresh and dry root weight, leaf number, total leaf area, and stomatal conductance. Stressed plants, however, increased their water-use efficiency and leaf soluble sugar concentration, decreased the shoot-to-root ratio and lowered their osmotic potential. Despite a decrease in photosynthetic pigments, the photosynthesis light phase remained unaffected under water stress. Water stress increased sucrose phosphate synthase activity in the leaves but not in the roots. Water stress inhibited sucrose:sucrose 1-fructosyltransferase and fructan:fructan 1 fructosyltransferase after 19 weeks of culture and slightly increased fructan 1-exohydrolase activity. The root inulin concentration, expressed on a dry-weight basis, and the mean degree of polymerization of the inulin chain remained unaffected by water stress. Root chicory displayed resistance to water stress, but that resistance was obtained at the expense of growth, which in turn led to a significant decrease in inulin production.

A Simple and Accurate Method for Determining Wheat Grain Fructan Content and Average Degree of Polymerization

An improved method for the measurement of fructans in wheat grains is presented. A mild acid treatment is used for fructan hydrolysis, followed by analysis of the released glucose and fructose with high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Not only the amount of fructose set free from fructans but also the released glucose can be quantified accurately, allowing determination of the average degree of polymerization of fructans (DP(av)). Application of the mild acid treatment to different grain samples demonstrated that a correction should be made for the presence of sucrose and raffinose, but not for stachyose or higher raffinose oligosaccharides. The fructan content and DP(av) of spelt flour, wheat flour, and whole wheat flour were 0.6%, 1.2%, and 1.8% of the total weight and 4, 5, and 6, respectively. Validation experiments demonstrate that the proposed quantification method is accurate and repeatable and that also the DP(av) determination is precise.

Comparison of fructan dynamics in two wheat cultivars with different capacities of accumulation and remobilization under drought stress

Remobilization of stored carbohydrates in the stem of wheat plants is an important contributor to grain filling under drought stress (DS) conditions. A massive screening on Iranian wheat cultivars was performed based on stem dry weight changes under well-watered and DS conditions. Two cultivars, Shole and Crossed Falat Hamun (CFH), with different fructan accumulation and remobilization behavior were selected for further studies. Water-soluble carbohydrates (WSCs) and fructan metabolizing enzymes were studied both in the stem penultimate and in sucrose (Suc) treated, excised leaves. Under drought, CFH produced higher grain yields than Shole (412 vs 220 g m(-2)). Also, grain yield loss under drought was more limited in CFH than in Shole (17 vs 54%). Under drought, CFH accumulated more graminan-type fructo-oligosaccharides than Shole. After anthesis, fructan 6-exohydrolase (6-FEH; EC 3.2.1.154) activities increased more prominently than fructan 1-exohydrolase (EC 3.2.1.153) activities during carbon remobilization. Interestingly, CFH showed higher 6-FEH activities in the penultimate than Shole. The field experiment results suggest that the combined higher remobilization efficiency and high 6-FEH activities in stems of wheat could contribute to grain yield under terminal drought. Similar to the penultimate, fructan metabolism differed strongly in Suc-treated detached leaves of selected cultivars. This suggests that variation in the stem fructan among wheat cultivars grown in the field could be traced by leaf blade induction experiments.

Understanding the Role of Defective Invertases in Plants: Tobacco Nin88 Fails to Degrade Sucrose

An inactive invertase may indirectly stimulate the activity of active cell wall invertases. Cell wall invertases (cwINVs), with a high affinity for the cell wall, are fundamental enzymes in the control of plant growth, development, and carbon partitioning. Most interestingly, defective cwINVs have been described in several plant species. Their highly attenuated sucrose (Suc)-hydrolyzing capacity is due to the absence of aspartate-239 (Asp-239) and tryptophan-47 (Trp-47) homologs, crucial players for stable binding in the active site and subsequent hydrolysis. However, so far, the precise roles of such defective cwINVs remain unclear. In this paper, we report on the functional characterization of tobacco (Nicotiana tabacum) Nin88, a presumed fully active cwINV playing a crucial role during pollen development. It is demonstrated here that Nin88, lacking both Asp-239 and Trp-47 homologs, has no invertase activity. This was further supported by modeling studies and site-directed mutagenesis experiments, introducing both Asp-239 and Trp-47 homologs, leading to an enzyme with a distinct Suc-hydrolyzing capacity. In vitro experiments suggest that the addition of Nin88 counteracts the unproductive and rather aspecific binding of tobacco cwINV1 to the wall, leading to higher activities in the presence of Suc and a more efficient interaction with its cell wall inhibitor. A working model is presented based on these findings, allowing speculation on the putative role of Nin88 in muro. The results presented in this work are an important first step toward unraveling the specific roles of plant defective cwINVs.

Fructan Metabolism in Developing Wheat (Triticum aestivum L.) Kernels

Although fructans play a crucial role in wheat kernel development, their metabolism during kernel maturation is far from being understood. In this study, all major fructan-metabolizing enzymes together with fructan content, fructan degree of polymerization and the presence of fructan oligosaccharides were examined in developing wheat kernels (Triticum aestivum L. var. Homeros) from anthesis until maturity. Fructan accumulation occurred mainly in the first 2 weeks after anthesis, and a maximal fructan concentration of 2.5 ± 0.3 mg fructan per kernel was reached at 16 days after anthesis (DAA). Fructan synthesis was catalyzed by 1-SST (sucrose:sucrose 1-fructosyltransferase) and 6-SFT (sucrose:fructan 6-fructosyltransferase), and to a lesser extent by 1-FFT (fructan:fructan 1-fructosyltransferase). Despite the presence of 6G-kestotriose in wheat kernel extracts, the measured 6G-FFT (fructan:fructan 6G-fructosyltransferase) activity levels were low. During kernel filling, which lasted from 2 to 6 weeks after anthesis, kernel fructan content decreased from 2.5 ± 0.3 to 1.31 ± 0.12 mg fructan per kernel (42 DAA) and the average fructan degree of polymerization decreased from 7.3 ± 0.4 (14 DAA) to 4.4 ± 0.1 (42 DAA). FEH (fructan exohydrolase) reached maximal activity between 20 and 28 DAA. No fructan-metabolizing enzyme activities were registered during the final phase of kernel maturation, and fructan content and structure remained unchanged. This study provides insight into the complex metabolism of fructans during wheat kernel development and relates fructan turnover to the general phases of kernel development.

A wheat 1‐FEH w3 variant underlies enzyme activity for stem WSC remobilization to grain under drought

In wheat stems, the levels of fructan-dominated water-soluble carbohydrates (WSC) do not always correlate well with grain yield. Field drought experiments were carried out to further explain this lack of correlation. Wheat (Triticum aestivum) varieties, Westonia, Kauz and c. 20 genetically diverse double haploid (DH) lines derived from them were investigated. Substantial genotypic differences in fructan remobilization were found and the 1-FEH w3 gene was shown to be the major contributor in the stem fructan remobilization process based on enzyme activity and gene expression results. A single nucleotide polymorphism (SNP) was detected in an auxin response element in the 1-FEH w3 promoter region, therefore we speculated that the mutated Westonia allele might affect gene expression and enzyme activity levels. A cleaved amplified polymorphic (CAP) marker was generated from the SNP. The harvested results showed that the mutated Westonia 1-FEH w3 allele was associated with a higher thousand grain weight (TGW) under drought conditions in 2011 and 2012. These results indicated that higher gene expression of 1-FEH w3 and 1-FEH w3 mediated enzyme activities that favoured stem WSC remobilization to the grains. The CAP marker residing in the 1-FEH w3 promoter region may facilitate wheat breeding by selecting lines with high stem fructan remobilization capacity under terminal drought.

Crystal structure of 6-SST/6-SFT from Pachysandra terminalis, a plant fructan biosynthesizing enzyme in complex with its acceptor substrate 6-kestose

Fructans play important roles as reserve carbohydrates and stress protectants in plants, and additionally serve as prebiotics with emerging antioxidant properties. Various fructan types are synthesized by an array of plant fructosyltransferases belonging to family 32 of the glycoside hydrolases (GH32), clustering together with GH68 in Clan-J. Here, the 3D structure of a plant fructosyltransferase from a native source, the Pachysandra terminalis 6-SST/6-SFT (Pt6-SST/6-SFT), is reported. In addition to its 1-SST (1-kestose-forming) and hydrolytic side activities, the enzyme uses sucrose to create graminan- and levan-type fructans, which are probably associated with cold tolerance in this species. Furthermore, a Pt6-SST/6-SFT complex with 6-kestose was generated, representing a genuine acceptor binding modus at the +1, +2 and +3 subsites in the active site. The enzyme shows a unique configuration in the vicinity of its active site, including a unique D/Q couple located at the +1 subsite that plays a dual role in donor and acceptor substrate binding. Furthermore, it shows a unique orientation of some hydrophobic residues, probably contributing to its specific functionality. A model is presented showing formation of a β(2-6) fructosyl linkage on 6-kestose to create 6,6-nystose, a mechanism that differs from the creation of a β(2-1) fructosyl linkage on sucrose to produce 1-kestose. The structures shed light on the evolution of plant fructosyltransferases from their vacuolar invertase ancestors, and contribute to further understanding of the complex structure-function relationships within plant GH32 members.

Sink filling, inulin metabolizing enzymes and carbohydrate status in field grown chicory (Cichorium intybus L.)

Inulin is a fructose-based polymer that is isolated from chicory (Cichorium intybus L.) taproots. The degree of polymerization (DP) determines its application and hence the value of the crop. The DP is highly dependent on the field conditions and harvest time. Therefore, the present study was carried out with the objective to understand the regulation of inulin metabolism and the process that determines the chain length and inulin yield throughout the whole growing season. Metabolic aspects of inulin production and degradation in chicory were monitored in the field and under controlled conditions. The following characteristics were determined in taproots: concentrations of glucose, fructose and sucrose, the inulin mean polymer length (mDP), yield, gene expression and activity of enzymes involved in inulin metabolism. Inulin synthesis, catalyzed by sucrose:sucrose 1-fructosyltransferase (EC 2.4.1.99) (1-SST) and fructan:fructan 1-fructosyltransferase (EC 2.4.1.100) (1-FFT), started at the onset of taproot development. Inulin yield as a function of time followed a sigmoid curve reaching a maximum in November. Inulin reached a maximum mDP of about 15 in September, than gradually decreased. Based on the changes observed in the pattern of inulin accumulation, we defined three different phases in the growing season and analyzed product formation, enzyme activity and gene expression in these defined periods. The results were validated by performing experiments under controlled conditions in climate rooms. Our results show that the decrease in 1-SST that starts in June is not regulated by day length and temperature. From mid-September onwards, the mean degree of polymerization (mDP) decreased gradually although inulin yield still increased. The decrease in mDP combined with increased yield results from fructan exohydrolase activity, induced by low temperature, and the back transfer activity of 1-FFT. Overall, this study provides background information on how to improve inulin yield and quality in chicory.