Potential of fructans as natural cryoprotectant agents in plant cryopreservation: concept validation on Arabidopsis thaliana L.

BACKGROUND: Today, synthetic chemicals are used in vitrification solutions for cryopreservation studies to mimic natural cryoprotectants that supply tolerance to organisms in nature against freezing stress. In the case of plants, PVS2, containing glycerol, dimethyl sulfoxide (Me2SO), ethylene glycol and sucrose, is considered as the golden standard for successful cryopreservation. However, Me2SO can generally cause toxicity to certain plant cells, adversely affecting viability after freezing and/or thawing. Hence, the replacement (or substantial reduction) of Me2SO by cheap, non-toxic and natural cryoprotectants became a matter of high priority to vitrification solutions or reducing their content gained escalating importance for the cryopreservation of plants. Fructans, sucrose derivatives mainly consisting of fructose residues, are candidate cryoprotectants. OBJECTIVE: Inspired by their protective role in nature, we here explored, for the first time, the potential of an array of 8 structurally different fructans as cryoprotectants in plant cryopreservation. MATERIALS AND METHODS: Arabidopsis thaliana L. seedlings were used as a model system with a one-step vitrification method. PVS2 solutions with different Me2SO and fructan contents were evaluated. RESULTS: It was found that branched low DP graminan, extracted from milky stage wheat kernels, led to the highest recovery (85%) among tested fructans with 12.5% Me2SO after cryopreservation, which was remarkably close to the viability (90%) observed with the original PVS2 containing 15% Me2SO. Moreover, its protective efficacy could be further optimized by addition of vitamin C acting as an antioxidant. CONCLUSION: Such novel formulations offer great perspectives for cryopreservation of various crop species. Doi.org/10.54680/fr24410110512.

Nectar traits differ between pollination syndromes in Balsaminaceae.

BACKGROUND AND AIMS: The attractiveness of nectar rewards depends both on the quantity of nectar produced and on its chemical composition. It is known that nectar quantity and chemical composition can differ in plant species depending on the main pollinator associated with the species. The main aims of this study were to test formally whether nectar traits are adapted to pollination syndromes in the speciose Balsaminaceae and, if so, whether a combination of nectar traits mirrors pollination syndromes. METHODS: Comparative methods based on Ornstein-Uhlenbeck models were used to test whether nectar volume, nectar sucrose proportion, sugar and amino acid concentration and amino acid composition had evolved as a function of pollination syndromes in 57 species of Balsaminaceae. Cluster analysis and ordination were performed to derive clusters of species resembling each other in nectar composition. KEY RESULTS: Evolutionary models for nectar volume and nectar sucrose proportion performed best when including information on pollination syndrome, while including such information improve model fit neither for sugar and amino acid concentration nor for amino acid composition. A significant relationship emerged between pollination syndrome and the combined nectar traits. CONCLUSIONS: Our results show that nectar volume and nectar sucrose proportion evolve rapidly towards optimal values associated with different pollination syndromes. The detection of a signal indicating that nectar traits in combination are to a certain extent able to predict pollination syndromes in Balsaminaceae suggests that a holistic approach including the whole set of nectar traits helps us to better understand evolution of nectar composition in response to pollinators.

Prebiotics to fight diseases: reality or fiction?

Bacteria living in the gastrointestinal tract are crucial for human health and disease occurrence. Increasing the beneficial intestinal microflora by consumption of prebiotics, which are 'functional foods', could be an elegant way to limit the number and incidence of disorders and to recover from dysbiosis or antibiotic treatments. This review focuses on the short-chain low-digestible carbohydrates (LDCs) which are metabolized by gut microbiota serving as energy source, immune system enhancers or facilitators of mineral uptake. Intake of foods containing LDCs can improve the state of health and may prevent diseases as for example certain forms of cancer. Given the large number of different molecules belonging to LDCs, we focused our attention on fructans (inulin, fructo-oligosaccharides), galacto-oligosaccharides and resistant starches and their therapeutic and protective applications. Evidence is accumulating that LDCs can inhibit bacterial and viral infections by modulating host defense responses and by changing the interactions between pathogenic and beneficial bacteria. Animal studies and studies on small groups of human subjects suggest that LDCs might help to counteract colorectal cancer, diabetes and metabolic syndrome. The action mechanisms of LDCs in the human body might be broader than originally thought, perhaps also including reactive oxygen species scavenging and signaling events.

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.

Large interclone differences in melezitose secretion in the facultatively ant-tended black bean aphid Aphis fabae.

Many aphids are known to engage in a trophic mutualism with ants, whereby the aphids secrete sugary-rich honeydew which is collected by the ants for food, and the ants, in exchange, protect the aphids against natural enemies. Previous results, however, suggest that the production of some of the honeydew sugars, such as the ant-attractant trisaccharide melezitose, may induce an indirect cost to the aphids. This led us to believe that large differences in the nature of the secreted honeydew might exist, due to some clones capitalizing more or less on their mutualistic interaction with ants, or due to some "cheater" clones foregoing the production of particular sugars, instead taking advantage of the ant-attracting effect of other non sugar-deficient clones, co-occurring on the same plant. Here we present data on clonal variation in the composition of honeydew of the black bean aphid Aphis fabae which confirm this prediction. In particular, our results show that there was large interclone variation in the amount of glucose, melezitose and total sugar produced. The variation in the production of melezitose, however, showed particularly large differences, with 54% (7 out of 13) of the clones screened being virtually deficient for the production of this sugar, irrespective of whether the aphid colonies were ant-tended or not. The consequences of this finding in the context of the evolution and maintenance of the ant-aphid mutualism, as well as the adaptive benefits of oligosaccharide synthesis in aphids and other insects are discussed.

Sugar ratios, glutathione redox status and phenols in the resurrection species Haberlea rhodopensis and the closely related non-resurrection species Chirita eberhardtii.

Because of their unique tolerance to desiccation, the so-called resurrection plants can be considered as excellent models for extensive research on plant reactions to environmental stresses. The vegetative tissues of these species are able to withstand long dry periods and to recover very rapidly upon re-watering. This study follows the dynamics of key components involved in leaf tissue antioxidant systems under desiccation in the resurrection plant Haberlea rhodopensis and the related non-resurrection species Chirita eberhardtii. In H. rhodopensis these parameters were also followed during recovery after full drying. A well-defined test system was developed to characterise the different responses of the two species under drought stress. Results show that levels of H2O2 decreased significantly both in H. rhodopensis and C. eberhardtii, but that accumulation of malondialdehyde was much more pronounced in the desiccation-tolerant H. rhodopensis than in the non-resurrection C. eberhardtii. A putative protective role could be attributed to accumulation of total phenols in H. rhodopensis during the late stages of drying. The total glutathione concentration and GSSG/GSH ratio increased upon complete dehydration of H. rhodopensis. Our data on soluble sugars suggest that sugar ratios might be important for plant desiccation tolerance. An array of different adaptations could thus be responsible for the resurrection phenotype of H. rhodopensis.

Induction of 1-FEH in mature chicory roots appears to be related to low temperatures rather than to leaf damage.

Large-scale inulin production from chicory roots (Cichorium intybus L.) is hampered by the induction of 1-FEH activity (fructan 1-exohydrolase) and concomitant fructose production in autumn, coincident with a period with low night temperatures that cause leaf damage. To understand whether leaf damage per se is sufficient for 1-FEH induction and fructan breakdown, we defoliated mature chicory plants at a preharvest stage (September 10) and investigated the changes in carbohydrate levels and 1-FEH activities. Also, the activities of 1-SST (sucrose:sucrose 1-fructosyl transferase, EC 2.4.1.99), 1-FFT (fructan:fructan 1-fructosyl transferase, EC 2.4.1.100), and acid invertase (EC 3.2.1.26) were determined. Defoliation did not result in a prompt fructan breakdown and increase in 1-FEH activity, but after 10 days fructan breakdown and 1-FEH activities became higher in the defoliated plants. Defoliation resulted in a sharp decrease in 1-SST activity over the first 24 h. Afterwards, root 1-SST activities of defoliated plants remained at a lower level than in control plants. 1-FFT and invertase activities were not affected by defoliation. It can be concluded that defoliation of plants at the preharvest stage by itself did not induce the same rapid changes as observed in naturally induced October roots by low temperature (harvest stage). Taken together with our finding that 1-FEH is not induced in chicory roots when plants are transferred to the greenhouse early autumn (minimal temperature 14 degrees C), we conclude that low temperatures might be essential for 1-FEH induction.

Defoliation induces fructan 1-exohydrolase II in Witloof chicory roots. Cloning and purification of two isoforms, fructan 1-exohydrolase IIa and fructan 1-exohydrolase IIb. Mass fingerprint of the fructan 1-exohydrolase II enzymes.

The cloning of two highly homologous chicory (Cichorium intybus var. foliosum cv Flash) fructan 1-exohydrolase cDNAs (1-FEH IIa and 1-FEH IIb) is described. Both isoenzymes could be purified from forced chicory roots as well as from the etiolated "Belgian endive" leaves where the 1-FEH IIa isoform is present in higher concentrations. Full-length cDNAs were obtained by a combination of reverse transcriptase-polymerase chain reaction (PCR), PCR and 5'- and 3'-rapid amplification of cDNA ends using primers based on N-terminal and conserved amino acid sequences. 1-FEH IIa and 1-FEH IIb cDNA-derived amino acid sequences are most homologous to a new group of plant glycosyl hydrolases harboring cell wall-type enzymes with acid isoelectric points. Unlike the observed expression profiles of chicory 1-FEH I, northern analysis revealed that 1-FEH II is expressed when young chicory plants are defoliated, suggesting that this enzyme can be induced at any developmental stage when large energy supplies are necessary (regrowth after defoliation).

Sucrose assimilation during early developmental stages of chicory (Cichorium intybus L.) plants.

The activities of enzymes of both sucrose and fructan metabolism were measured in chicory (Cichorium intybus L. cv. Turbo) plants during early vegetative growth. From 21 to 42 d after sowing (phase I), carbohydrates were used for structural growth and sucrose was predominantly cleaved by acid invertase whereas neutral invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13) activities were low. From 49 to 63 d after sowing (phase II) a cambium formed producing secondary tissues, concomitant with induced sucrose:sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99) and fructan:fructan-1-fructosyl transferase (EC 2.4.1.100) activities, and fructan synthesis in the roots. Accumulation of 1-SST mRNA occurred at the onset of thickening, indicating that 1-SST is controlled at a transcriptional level. Acid invertase activity gradually increased during phase I and remained high during early phase II. It subsequently decreased. The pattern of invertase mRNA accumulation correlated with the enzyme activities, indicating that acid invertase is controlled at the transcriptional level. Both acid invertase and 1-SST probably contributed to the sink strength in the root at the beginning of phase II.

Cloning and functional analysis of chicory root fructan1-exohydrolase I (1-FEH I): a vacuolar enzyme derivedfrom a cell-wall invertase ancestor? Mass fingerprint of the 1-FEH I enzyme.

This paper describes the cloning and functional analysis of chicory (Cichorium intybus L.) fructan 1-exohydrolase I cDNA (1-FEH I). To our knowledge it is the first plant FEH cloned. Full-length cDNA was obtained by a combination of RT-PCR, 5' and 3' RACE using primers based on N-terminal and conserved amino acid sequences. Electrophoretically purified 1-FEH I enzyme was further analyzed by in-gel trypsin digestion followed by matrix-assisted laser desorption ionization and electrospray time-of-flight tandem mass spectrometry. Functionality of the cDNA was demonstrated by heterologous expression in potato tubers. 1-FEH I takes a new, distinct position in the phylogenetic tree of plant glycosyl hydrolases being more homologous to cell-wall invertases (44-53%) than to vacuolar invertases (38-41%) and fructosyl transferases (33-38%). The 1-FEH I enzyme could not be purified from the apoplastic fluid at significantly higher levels than can be explained by cellular leakage. These and other data suggest a vacuolar localization for 1-FEH I. Also, the pI of the enzyme (6.5) is lower than expected from a typical cell-wall invertase. Unlike plant fructosyl transferases that are believed to have evolved from a vacuolar invertase, 1-FEH I might have evolved from a cell-wall invertase-like ancestor gene that later obtained a vacuolar targeting signal. 1-FEH I mRNA quantities increase in the roots throughout autumn, and especially when roots are stored at low temperature.

Fructan accumulation induced by nitrogen deficiency in barley leaves correlates with the level of sucrose:fructan 6-fructosyltransferase mRNA.

Hydroponically cultivated barley plants were exposed to nitrogen (N)-deficiency followed by N-resupply. Metabolic and genetic regulation of fructan accumulation in the leaves were investigated. Fructan accumulated in barley leaves under N-deficiency was mobilized during N-resupply. The enhanced total activity of fructan-synthesizing enzymes, sucrose:sucrose 1-fructosyltransferase (EC 2.4.1.99) and sucrose:fructan 6-fructosyltransferase (6-SFT; EC 2.4. 1.10) caused by N-deficiency decreased with the mobilization of fructan during N-resupply. The activity of the barley fructan-degrading enzyme, fructan exohydrolyase (EC 3.2.1.80) was less affected by the N status. The low level of foliar soluble acid invertase activity under N-deficiency conditions was maintained during the commencement of N-resupply but increased subsequently. Further analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot and northern blot demonstrated that the fructan accumulation and the total activity of fructan-synthesizing enzymes correlated with the 6-SFT mRNA level. We suggest that the changes in fructan levels under N stress are intimately connected with the regulation of fructan synthetic rate which is mostly controlled by 6-SFT.

The role of fructan in flowering of Campanula rapunculoides.

Inulin type fructan was detected in all vegetative organs of Campanula rapunculoides L. plants. All flower parts contained fructan at some developmental stage. A steady decrease was found in sepals during development. Petals, however, stored fructan in the bud stage. A rapid breakdown during opening of the flower resulted in high concentrations of glucose and especially fructose that may contribute to the osmotic driving force involved in petal expansion. Before complete shrivelling, the hexoses were apparently exported from flower parts. Fructans were hydrolysed and exported from the stamen and style tissue upon flower opening. Similarly, the major fructan reserves in the ovary were broken down almost simultaneously with those in other flower parts. Hexoses did not reach high levels in the ovary, probably because they were rapidly metabolized and/or incorporated by developing seeds.

Cloning, developmental, and tissue-specific expression of sucrose:sucrose 1-fructosyl transferase from Taraxacum officinale. Fructan localization in roots.

Sucrose:sucrose 1-fructosyl transferase (1-SST) is the key enzyme initiating fructan synthesis in Asteraceae. Using reverse transcriptase-PCR, we isolated the cDNA for 1-SST from Taraxacum officinale. The cDNA-derived amino acid sequence showed very high homology to other Asteracean 1-SSTs (Cichorium intybus 86%, Cynara scolymus 82%, Helianthus tuberosus 80%), but homology to 1-SST from Allium cepa (46%) and Aspergillus foetidus (18%) was much lower. Fructan concentrations, 1-SST activities, 1-SST protein, and mRNA concentrations were compared in different organs during vegetative and generative development of T. officinale plants. Expression of 1-SST was abundant in young roots but very low in leaves. 1-SST was also expressed at the flowering stages in roots, stalks, and receptacles. A good correlation was found between northern and western blots showing transcriptional regulation of 1-SST. At the pre-flowering stage, 1-SST mRNA concentrations and 1-SST activities were higher in the root phloem than in the xylem, resulting in the higher fructan concentrations in the phloem. Fructan localization studies indicated that fructan is preferentially stored in phloem parenchyma cells in the vicinity of the secondary sieve tube elements. However, inulin-like crystals occasionally appeared in xylem vessels.

Drought induces fructan synthesis and 1-SST (sucrose:sucrose fructosyltransferase) in roots and leaves of chicory seedlings (Cichorium intybus L.).

Seeds of Cichorium intybus L. var. foliosum cv. Flash were sown in acid-washed vermiculite and grown in a controlled-environment growth chamber. After 1 month of growth, plantlets did not contain sucrose:sucrose 1-fructosyltransferase (1-SST), the key enzyme in fructan biosynthesis. No fructan could be observed. Some of the plants were submitted to drought for 2 weeks. Glucose, fructose and sucrose concentrations increased in roots and leaves of stressed plants and the fructan concentration in roots and leaves was ten times higher than in control plants. The onset of fructan synthesis coincided with the increase in 1-SST activity in roots. Expression of the 1-SST gene could be observed in roots and leaves of stressed plants.