Recent advances on bioactive compounds, biosynthesis mechanism, and physiological functions of Nelumbo nucifera.

Nelumbo nucifera Gaertn, commonly known as lotus, is a genus comprising perennial and rhizomatous aquatic plants, found throughout Asia and Australia. This review aimed to cover the biosynthesis of flavonoids, alkaloids, and lipids in plants and their types in different parts of lotus. This review also examined the physiological functions of bioactive compounds in lotus and the extracts from different organs of the lotus plant. The structures and identities of flavonoids, alkaloids, and lipids in different parts of lotus as well as their biosynthesis were illustrated and updated. In the traditional medicine systems and previous scientific studies, bioactive compounds and the extracts of lotus have been applied for treating inflammation, cancer, liver disease, Alzheimer's disease, etc. We suggest future studies to be focused on standardization of the extract of lotus, and their pharmacological mechanisms as drugs or functional foods. This review is important for the lotus-based food processing and application.

Whole grain-rich diet reduces body weight and systemic low-grade inflammation without inducing major changes of the gut microbiome: a randomised cross-over trial.

OBJECTIVE: To investigate whether a whole grain diet alters the gut microbiome and insulin sensitivity, as well as biomarkers of metabolic health and gut functionality. DESIGN: 60 Danish adults at risk of developing metabolic syndrome were included in a randomised cross-over trial with two 8-week dietary intervention periods comprising whole grain diet and refined grain diet, separated by a washout period of ≥6 weeks. The response to the interventions on the gut microbiome composition and insulin sensitivity as well on measures of glucose and lipid metabolism, gut functionality, inflammatory markers, anthropometry and urine metabolomics were assessed. RESULTS: 50 participants completed both periods with a whole grain intake of 179±50 g/day and 13±10 g/day in the whole grain and refined grain period, respectively. Compliance was confirmed by a difference in plasma alkylresorcinols (p<0.0001). Compared with refined grain, whole grain did not significantly alter glucose homeostasis and did not induce major changes in the faecal microbiome. Also, breath hydrogen levels, plasma short-chain fatty acids, intestinal integrity and intestinal transit time were not affected. The whole grain diet did, however, compared with the refined grain diet, decrease body weight (p<0.0001), serum inflammatory markers, interleukin (IL)-6 (p=0.009) and C-reactive protein (p=0.003). The reduction in body weight was consistent with a reduction in energy intake, and IL-6 reduction was associated with the amount of whole grain consumed, in particular with intake of rye. CONCLUSION: Compared with refined grain diet, whole grain diet did not alter insulin sensitivity and gut microbiome but reduced body weight and systemic low-grade inflammation. TRIAL REGISTRATION NUMBER: NCT01731366; Results.

The Role of Cysteine Residues in Redox Regulation and Protein Stability of Arabidopsis thaliana Starch Synthase 1.

Starch biosynthesis in Arabidopsis thaliana is strictly regulated. In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influences its catalytic function 2) that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated by the physiological redox transmitters thioredoxin f1 (Trxf1), thioredoxin m4 (Trxm4) and the bifunctional NADPH-dependent thioredoxin reductase C (NTRC). AtSS1 displayed an activity change within the physiologically relevant redox range, with a midpoint potential equal to -306 mV, suggesting that AtSS1 is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%). Michaelis-Menten kinetics and molecular modeling suggest that both cysteines play important roles in enzyme catalysis, namely, Cys545 is involved in ADP-glucose binding and Cys164 is involved in acceptor binding. All the other single mutants had essentially complete redox sensitivity (98-99%). In addition of being part of a redox directed activity "light switch", reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein prior to its transport into the plastid. Cys442 can play an important role in enzyme stability upon oxidation. The physiological and phylogenetic relevance of these findings is discussed.

Virus-induced multiple gene silencing to study redundant metabolic pathways in plants: silencing the starch degradation pathway in Nicotiana benthamiana.

Virus-induced gene silencing (VIGS) is a rapid technique that allows for specific and reproducible post-transcriptional degradation of targeted mRNA. The method has been proven efficient for suppression of expression of many single enzymes. The metabolic networks of plants, however, often contain isoenzymes and gene families that are able to compensate for a mutation and mask the development of a silencing phenotype. Here, we show the application of multiple gene VIGS repression for the study of these redundant biological pathways. Several genes in the starch degradation pathway [disproportionating enzyme 1; (DPE1), disproportionating enzyme 2 (DPE2), and GWD] were silenced. The functionally distinct DPE enzymes are present in alternate routes for sugar export to the cytoplasm and result in an increase in starch production when silenced individually. Simultaneous silencing of DPE1 and DPE2 in Nicotiana benthamiana resulted in a near complete suppression in starch and accumulation of malto-oligosaccharides.

Starch-binding domains in the CBM45 family--low-affinity domains from glucan, water dikinase and α-amylase involved in plastidial starch metabolism.

Starch-binding domains are noncatalytic carbohydrate-binding modules that mediate binding to granular starch. The starch-binding domains from the carbohydrate-binding module family 45 (CBM45, http://www.cazy.org) are found as N-terminal tandem repeats in a small number of enzymes, primarily from photosynthesizing organisms. Isolated domains from representatives of each of the two classes of enzyme carrying CBM45-type domains, the Solanum tuberosumα-glucan, water dikinase and the Arabidopsis thaliana plastidial α-amylase 3, were expressed as recombinant proteins and characterized. Differential scanning calorimetry was used to verify the conformational integrity of an isolated CBM45 domain, revealing a surprisingly high thermal stability (T(m) of 84.8 °C). The functionality of CBM45 was demonstrated in planta by yellow/green fluorescent protein fusions and transient expression in tobacco leaves. Affinities for starch and soluble cyclodextrin starch mimics were measured by adsorption assays, surface plasmon resonance and isothermal titration calorimetry analyses. The data indicate that CBM45 binds with an affinity of about two orders of magnitude lower than the classical starch-binding domains from extracellular microbial amylolytic enzymes. This suggests that low-affinity starch-binding domains are a recurring feature in plastidial starch metabolism, and supports the hypothesis that reversible binding, effectuated through low-affinity interaction with starch granules, facilitates dynamic regulation of enzyme activities and, hence, of starch metabolism.

Repression of both isoforms of disproportionating enzyme leads to higher malto-oligosaccharide content and reduced growth in potato.

Two glucanotransferases, disproportionating enzyme 1 (StDPE1) and disproportionating enzyme 2 (StDPE2), were repressed using RNA interference technology in potato, leading to plants repressed in either isoform individually, or both simultaneously. This is the first detailed report of their combined repression. Plants lacking StDPE1 accumulated slightly more starch in their leaves than control plants and high levels of maltotriose, while those lacking StDPE2 contained maltose and large amounts of starch. Plants repressed in both isoforms accumulated similar amounts of starch to those lacking StDPE2. In addition, they contained a range of malto-oligosaccharides from maltose to maltoheptaose. Plants repressed in both isoforms had chlorotic leaves and did not grow as well as either the controls or lines where only one of the isoforms was repressed. Examination of photosynthetic parameters suggested that this was most likely due to a decrease in carbon assimilation. The subcellular localisation of StDPE2 was re-addressed in parallel with DPE2 from Arabidopsis thaliana by transient expression of yellow fluorescent protein fusions in tobacco. No translocation to the chloroplasts was observed for any of the fusion proteins, supporting a cytosolic role of the StDPE2 enzyme in leaf starch metabolism, as has been observed for Arabidopsis DPE2. It is concluded that StDPE1 and StDPE2 have individual essential roles in starch metabolism in potato and consequently repression of these disables regulation of leaf malto-oligosaccharides, starch content and photosynthetic activity and thereby plant growth possibly by a negative feedback mechanism.

Effects of beta-1,3-glucan from Septoria tritici on structural defence responses in wheat.

The accumulation of the pathogenesis-related (PR) proteins beta-1,3-glucanase and chitinase and structural defence responses were studied in leaves of wheat either resistant or susceptible to the hemibiotrophic pathogen Septoria tritici. Resistance was associated with an early accumulation of beta-1,3-glucanase and chitinase transcripts followed by a subsequent reduction in level. Resistance was also associated with high activity of beta-1,3-glucanase, especially in the apoplastic fluid, in accordance with the biotrophic/endophytic lifestyle of the pathogen in the apoplastic spaces, thus showing the highly localized accumulation of defence proteins in the vicinity of the pathogen. Isoform analysis of beta-1,3-glucanase from the apoplastic fluid revealed that resistance was associated with the accumulation of an endo-beta-1,3-glucanase, previously implicated in defence against pathogens, and a protein with identity to ADPG pyrophosphatase (92%) and germin-like proteins (93%), which may be involved in cell wall reinforcement. In accordance with this, glycoproteins like extensin were released into the apoplast and callose accumulated to a greater extent in cell walls, whereas lignin and polyphenolics were not found to correlate with defence. Treatment of a susceptible wheat cultivar with purified beta-1,3-glucan fragments from cell walls of S. tritici gave complete protection against disease and this was accompanied by increased gene expression of beta-1,3-glucanase and the deposition of callose. Collectively, these data indicate that resistance is dependent on a fast, initial recognition of the pathogen, probably due to beta-1,3-glucan in the fungal cell walls, and this results in the accumulation of beta-1,3-glucanase and structural defence responses, which may directly inhibit the pathogen and protect the host against fungal enzymes and toxins.

A CBM20 low-affinity starch-binding domain from glucan, water dikinase.

The family 20 carbohydrate-binding module (CBM20) of the Arabidopsis starch phosphorylator glucan, water dikinase 3 (GWD3) was heterologously produced and its properties were compared to the CBM20 from a fungal glucoamylase (GA). The GWD3 CBM20 has 50-fold lower affinity for cyclodextrins than that from GA. Homology modelling identified possible structural elements responsible for this weak binding of the intracellular CBM20. Differential binding of fluorescein-labelled GWD3 and GA modules to starch granules in vitro was demonstrated by confocal laser scanning microscopy and yellow fluorescent protein-tagged GWD3 CBM20 expressed in tobacco confirmed binding to starch granules in planta.

Sulfur starvation in rice: the effect on photosynthesis, carbohydrate metabolism, and oxidative stress protective pathways.

Sulfur-deficient plants generate a lower yield and have a reduced nutritional value. The process of sulfur acquisition and assimilation play an integral role in plant metabolism, and response to sulfur deficiency involves a large number of plant constituents. Rice (Oryza sativa) is the second most consumed cereal grain, and the effects of sulfur deprivation in rice were analyzed by measuring changes in photosynthesis, carbohydrate metabolism, and antioxidants. The photosynthetic apparatus was severely affected under sulfur deficiency. The Chl content was reduced by 49% because of a general reduction of PSII and PSI and the associated light-harvesting antenna. The PSII efficiency was 31% lower at growth light, and the ability of PSI to photoreduce NADP+ was decreased by 61%. The Rubisco content was also significantly reduced in the sulfur-deprived plants. The imbalances between PSII and PSI, and between photosynthesis and carbon fixation led to a general over-reduction of the photosynthetic electron carriers (higher 1-q(P)). Chromatographic analysis showed that the level of monosaccharides was lower and starch content higher in the sulfur-deprived plants. In contrast, no changes in metabolite levels were found in the tricarboxylic acid or Calvin cycle. The level of the thiol-containing antioxidant, GSH, was 70% lower and the redox state was significantly more oxidized. These changes in GSH status led to an upregulation of the cytosolic isoforms of GSH reductase and monodehydroascorbate reductase. In addition, alternative antioxidants like flavonoids and anthocyanins were increased in the sulfur-deprived plants.

The amyloplast proteome of potato tuber.

Potato (Solanum tuberosum) is the fourth largest crop worldwide in yield, and cv. Kuras is the major starch potato of northern Europe. Storage starch is packed densely in tuber amyloplasts, which become starch granules. Amyloplasts of soil-grown mini-tubers and agar-grown micro-tubers of cv. Kuras were purified. The mini-tuber amyloplast preparation was enriched 10-20-fold and the micro-tuber amyloplast approximately fivefold over comparative total protein extracts. Proteins separated by SDS-PAGE were digested with trypsin, analysed by mass spectrometry and identified by mascot software searches against an in-house potato protein database and the NCBI non-redundant plant database. The differential growth conditions for mini- and micro-tubers gave rise to rather different protein profiles, but the major starch granule-bound proteins were identical for both and dominated by granule-bound starch synthase I, starch synthase II and alpha-glucan water dikinase. Soluble proteins were dominated by starch phosphorylase L-1, other large proteins of the classes 'starch and sucrose metabolism', 'pentose phosphate pathway', 'glycolysis', 'amino acid metabolism', and other proteins such as plastid chaperonins. The majority of the identified proteins had a predicted plastid transit peptide, supporting their presence in the amyloplast. However, several highly expressed proteins had no transit peptide, such as starch phosphorylase H, or had a predicted mitochondrial location. Intriguingly, all polyphenol oxidases, a family of enolases, one transketolase, sulfite reductase, deoxynucleoside kinase-like and dihydroxy-acid dehydrase had twin-arginine translocation motifs, and a homologue to dihydrolipoamide dehydrogenase had a Sec (secretory) motif; these motifs usually target thylakoid-like structures.

Starch phosphorylation: a new front line in starch research.

Starch is the primary energy reserve in higher plants and is, after cellulose, the second most abundant carbohydrate in the biosphere. It is also the most important energy source in the human diet and, being a biodegradable polymer with well-defined chemical properties, has an enormous potential as a versatile renewable resource. The only naturally occurring covalent modification of starch is phosphorylation. Starch phosphate esters were discovered a century ago but were long regarded as a curiosity, receiving little attention. Indeed, the mechanism for starch phosphorylation remained completely unknown until recently. The starch-phosphorylating enzyme is an alpha-glucan water dikinase. It is now known that starch phosphorylation plays a central role in starch metabolism.

Production of highly phosphorylated glycopolymers by expression of R1 in Escherichia coli.

The possible involvement of the starch bound R1 protein from potato (Solanum tuberosum L.) in the phosphorylation of starch was investigated by functional expression and characterisation of R1 in Escherichia coli. By expression of R1 in E. coli it is shown that it is possible to produce glycopolymers, e.g., glycogen, with an increased degree of phosphate substitution. The expression of R1 in E. coli resulted in a sixfold increase in glycogen bound phosphate and in an increased accumulation of glycogen leading to a glycogen excess (gex) phenotype. There was an overall shift in the unit-chain length of the isolated glycogen towards smaller degrees of polymerisation. The pleiotropic effects on the glycogen biosynthetic and amylolytic enzyme activities was investigated and showed an increase in ADPglucose pyrophosphorylase activity, as well as a decrease in exo-amylolytic activity. These results are discussed in relation to starch phosphorylation and a possible role of R1 in this respect.