Search Results (9)

Plants adapt to abiotic stresses by a variety of physiological and molecular mechanisms, among which the root plays important roles via responding to underground and soilborne signals. Fructan is a polysaccharide involved in energy metabolism and stress adaptation. Orobanche cumana is a holo-parasitic plant that mainly attaches to the root of the host sunflower (Helianthus annuus). Oc6-FEH, a fructan 6-exohydrolase from O. cumana, is involved in both fructan metabolism and flooding responses. Expression of Oc6-FEH is induced by flooding and indole-3-acetic acid (IAA). Oc6-FEH possesses fructan catabolism activity and is associated with fructose release. Overexpression of Oc6-FEH in the host sunflower reduces malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) accumulation, boosts the activities of antioxidant enzymes, including peroxidase (POD) and superoxide dismutase (SOD), and enhances photosynthetic performance. The expression level of Oc6-FEH was found to be positively associated with the flooding tolerance of invading O. cumana, which is connected to the host root. Furthermore, IAA treatment also improved the flooding tolerance of O. cumana. In summary, the metabolism of fructan and the activity of Oc6-FEH were demonstrated to ameliorate waterlogging stress. Oc6-FEH provides a promising genetic target for the improvement of flooding tolerance in crops. Full article

Lettuce is a globally cultivated and consumed leafy crop. Here we developed an efficient tobacco rattle virus (TRV)-based guide RNA (gRNA) delivery system for CRISPR/Cas editing in the commercial lettuce cultivar ‘Noga’. Plants stably expressing Cas9 were inoculated with TRV vectors carrying gRNAs targeting five nutrient-associated genes. The system achieved an average editing efficiency of 48.7%, with up to 78.9% of regenerated plantlets showing independent mutations. This approach eliminates the need for antibiotic selection, simplifying tissue culture processes. The system supports diverse applications, including Cas12a editing and large-fragment deletions using dual gRNA sets. Targeting the fructan 1-exohydrolase 2 (1-FEH2) gene produced knockout lines with significant increases in prebiotic dietary fibre fructan content, up to 5.2-fold, and an average rise in the degree of polymerisation by 2.15 units compared with controls. Combining 1-FEH1 and 1-FEH2 knockouts did not further increase fructan levels, revealing 1-FEH2 as the predominant isozyme in lettuce. RT-qPCR analysis showed reduced expression of the upstream biosynthetic enzyme sucrose:sucrose 1-fructosyl transferase (1-SST), suggesting potential feedback inhibition in fructan metabolism. This TRV-based gene editing approach, utilised here to increase fructan content, could be applied to improve other valuable traits in lettuce, and may inspire similar systems to enhance nutritional content of crops. Full article

Fructan 1-exohydrolase (1-FEH) is one of the major enzymes in water-soluble carbohydrate (WSC) remobilisation for grains in wheat. We investigated the functional role of 1-FEH w1, w2, and w3 isoforms in WSC remobilisation under post-anthesis water deficit using mutation lines derived from the Australian wheat variety Chara. F1 seeds, developed by backcrossing the 1-FEH w1, w2, and w3 mutation lines with Chara, were genotyped using the Infinium 90K SNP iSelect platform to characterise the mutated region. Putative deletions were identified in FEH mutation lines encompassing the FEH genomic regions. Mapping analysis demonstrated that mutations affected significantly longer regions than the target FEH gene regions. Functional roles of the non-target genes were carried out utilising bioinformatics and confirmed that the non-target genes were unlikely to confound the effects considered to be due to the influence of 1-FEH gene functions. Glasshouse experiments revealed that the 1-FEH w3 mutation line had a slower degradation and remobilisation of fructans than the 1-FEH w2 and w1 mutation lines and Chara, which reduced grain filling and grain yield. Thus, 1-FEH w3 plays a vital role in reducing yield loss under drought. This insight into the distinct role of the 1-FEH isoforms provides new gene targets for water-deficit-tolerant wheat breeding. Full article

As the main reserve carbohydrate in garlic, fructan contributes to garlic’s yield and quality formation. Numerous studies have shown that plant fructan metabolism induces a stress response to adverse environments. However, the transcriptional regulation mechanism of garlic fructan in low-temperature environments is still unknown. In this study, the fructan metabolism of garlic seedlings under low-temperature stress was revealed by transcriptome and metabolome approaches. With the extension of stress time, the number of differentially expressed genes and metabolites increased. Using weighted gene co-expression network analysis (WGCNA), three key enzyme genes related to fructan metabolism were screened (a total of 12 transcripts): sucrose: sucrose 1-fructosyltransferase (1-SST) gene; fructan: fructan 6G fructosyltransferase (6G-FFT) gene; and fructan 1-exohydrolase (1-FEH) gene. Finally, two hub genes were obtained, namely Cluster-4573.161559 (6G-FFT) and Cluster-4573.153574 (1-FEH). The correlation network and metabolic heat map analysis between fructan genes and carbohydrate metabolites indicate that the expression of key enzyme genes in fructan metabolism plays a positive promoting role in the fructan response to low temperatures in garlic. The number of genes associated with the key enzyme of fructan metabolism in trehalose 6-phosphate was the highest, and the accumulation of trehalose 6-phosphate content may mainly depend on the key enzyme genes of fructan metabolism rather than the enzyme genes in its own synthesis pathway. This study not only obtained the key genes of fructan metabolism in garlic seedlings responding to low temperatures but also preliminarily analyzed its regulatory mechanism, providing an important theoretical basis for further elucidating the cold resistance mechanism of garlic fructan metabolism. Full article

Plant Glycoside Hydrolase Family 32 (PGHF32) contains the fructosyltransferases and fructan exohydrolase enzymes responsible for fructan metabolism, in addition to closely related vacuolar and cell wall acid invertases. Agave species produce complex and dynamic fructan molecules (agavins) requiring 4 different fructosyltransferase activities (1-SST, 1-FFT, 6G-FFT and 6-SFT) for their synthesis. Combined analysis of RNAseq and genome data for A. tequilana led to the characterization of the genes encoding 3 fructosyltransferases for this species and support the hypothesis that no separate 6-SFT type enzyme exists in A. tequilana, suggesting that at least one of the fructosyltransferases identified may have multiple enzymatic activities. Structures for PGHF32 genes varied for A. tequilana and between other plant species but were conserved for different enzyme types within a species. The observed patterns are consistent with the formation of distinct gene structures by intron loss. Promoter analysis of the PGHF32 genes identified abundant putative regulatory motifs for light regulation and tissue-specific expression, and these regulatory mechanisms were confirmed experimentally for leaf tissue. Motifs for phytohormone response, carbohydrate metabolism and dehydration responses were also uncovered. Based on the regulatory motifs, full-length cDNAs for MYB, GATA, DOF and GBF transcription factors were identified and their phylogenetic distribution determined by comparison with other plant species. In silico expression analysis for the selected transcription factors revealed both tissue-specific and developmental patterns of expression, allowing candidates to be identified for detailed analysis of the regulation of fructan metabolism in A. tequilana at the molecular level. Full article

Enzymes with fructan exohydrolase (FEH) activity are present not only in fructan-synthesizing species but also in non-fructan plants. This has led to speculation about their functions in non-fructan species. Here, a cell wall invertase-related Zm-6&1-FEH2 with no “classical” invertase motif was identified in maize. Following heterologous expression in Pichia pastoris and in Nicotiana benthamiana leaves, the enzyme activity of recombinant Zm-6&1-FEH2 displays substrate specificity with respect to inulin and levan. Subcellular localization showed Zm-6&1-FEH2 exclusively localized in the apoplast, and its expression profile was strongly dependent on plant development and in response to drought and abscisic acid. Furthermore, formation of 1-kestotriose, an oligofructan, was detected in vivo and in vitro and could be hydrolyzed by Zm-6&1-FEH2. In summary, these results support that Zm-6&1-FEH2 enzyme from maize can degrade both inulin-type and levan-type fructans, and the implications of the co-existence of Zm-6&1-FEH2 and 1-kestotriose are discussed. Full article

In northern regions, annual and perennial overwintering plants such as wheat and temperate grasses accumulate fructan in vegetative tissues as an energy source. This is necessary for the survival of wintering tissues and degrading fructan for regeneration in spring. Other types of wintering plants, including chicory and asparagus, store fructan as a reserve carbohydrate in their roots during winter for shoot- and spear-sprouting in spring. In this review, fructan metabolism in plants during winter is discussed, with a focus on the fructan-degrading enzyme, fructan exohydrolase (FEH). Plant fructan synthase genes were isolated in the 2000s, and FEH genes have been isolated since the cloning of synthase genes. There are many types of FEH in plants with complex-structured fructan, and these FEHs control various kinds of fructan metabolism in growth and survival by different physiological responses. The results of recent studies on the fructan metabolism of plants in winter have shown that changes in fructan contents in wintering plants that are involved in freezing tolerance and snow mold resistance might be largely controlled by regulation of the expressions of genes for fructan synthesis, whereas fructan degradation by FEHs is related to constant energy consumption for survival during winter and rapid sugar supply for regeneration or sprouting of tissues in spring. Full article

About 15% of higher plants have acquired the ability to convert sucrose into fructans. Fructan degradation is catalyzed by fructan exohydrolases (FEHs), which are structurally related to cell wall invertases (CWI). However, the biological function(s) of FEH enzymes in non-fructan species have remained largely enigmatic. In the present study, one maize CWI-related enzyme named Zm-6&1-FEH1, displaying FEH activity, was explored with respect to its substrate specificities, its expression during plant development, and its possible interaction with CWI inhibitor protein. Following heterologous expression in Pichia pastoris and in N. benthamiana leaves, recombinant Zm-6&1-FEH1 revealed substrate specificities of levan and inulin, and also displayed partially invertase activity. Expression of Zm-6&1-FEH1 as monitored by qPCR was strongly dependent on plant development and was further modulated by abiotic stress. To explore whether maize FEH can interact with invertase inhibitor protein, Zm-6&1-FEH1 and maize invertase inhibitor Zm-INVINH1 were co-expressed in N. benthamiana leaves. Bimolecular fluorescence complementation (BiFC) analysis and in vitro enzyme inhibition assays indicated productive complex formation. In summary, the results provide support to the hypothesis that in non-fructan species FEH enzymes may modulate the regulation of CWIs. Full article

Jerusalem artichoke tubers have diverse applications in the food industry as well as in biotechnology. Their suitability depends mostly on the inulin content. Seasonal fluctuations of fructan exohydrolase activity responsible for inulin degradation was investigated in the tubers of three Jerusalem artichoke cultivars. The changes of fructan exohydrolase activity positively correlated with the changes of the content of total and short fructooligosaccharides. Therefore, to extract inulin with higher degree of polymerization for biotechnological purposes, the tubers of Jerusalem artichoke should be uprooted in autumn before the level of fructan exohydrolase reaches its maximum. If short fructooligosaccharides are desirable, the tubers in late autumn or spring tubers overwintered in soil are suitable. Full article