Celiac disease: in vitro and in vivo safety and palatability of wheat-free sorghum food products.

BACKGROUND & AIMS: Celiac disease is a condition in which genetically predisposed people have an autoimmune reaction to gluten proteins found in all wheat types and closely related cereals such as barley and rye. This reaction causes the formation of autoantibodies and the destruction of the villi in the small intestine, which results in malabsorption of nutrientsand other gluten-induced autoimmune diseases. Sorghum is a cereal grain with potential to be developed into an important crop for human food products. The flour produced from white sorghum hybrids is light in color and has a bland, neutral taste that does not impart unusual colors or flavors to food products. These attributes make it desirable for use in wheat-free food products. While sorghum is considered as a safe food for celiac patients, primarily due to its relationship to maize, no direct testing has been conducted on its safety for gluten intolerance. Therefore studies are needed to assess its safety and tolerability in celiac patients. Thus the aim of the present study was to assess safety and tolerability of sorghum flour products in adult celiac disease patients, utilizing an in vitro and in vivo challenge. RESULTS: Sorghum protein digests did not elicit any morphometric or immunomediated alteration of duodenal explants from celiac patients. Patients fed daily for 5 days with sorghum-derived food product did not experience gastrointestinal or non-gastrointestinal symptoms and the level of anti-transglutaminase antibodies was unmodified at the end of the 5-days challenge. CONCLUSIONS: Sorghum-derived products did not show toxicity for celiac patients in both in vitro and in vivo challenge. Therefore sorghum can be considered safe for people with celiac disease.

Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria.

Although plant cell bioenergetics is strongly affected by abiotic stresses, mitochondrial metabolism under stress is still largely unknown. Interestingly, plant mitochondria may control reactive oxygen species (ROS) generation by means of energy-dissipating systems. Therefore, mitochondria may play a central role in cell adaptation to abiotic stresses, which are known to induce oxidative stress at cellular level. With this in mind, in recent years, studies have been focused on mitochondria from durum wheat, a species well adapted to drought stress. Durum wheat mitochondria possess three energy-dissipating systems: the ATP-sensitive plant mitochondrial potassium channel (PmitoK(ATP)); the plant uncoupling protein (PUCP); and the alternative oxidase (AOX). It has been shown that these systems are able to dampen mitochondrial ROS production; surprisingly, PmitoK(ATP) and PUCP (but not AOX) are activated by ROS. This was found to occur in mitochondria from both control and hyperosmotic-stressed seedlings. Therefore, the hypothesis of a 'feed-back' mechanism operating under hyperosmotic/oxidative stress conditions was validated: stress conditions induce an increase in mitochondrial ROS production; ROS activate PmitoK(ATP) and PUCP that, in turn, dissipate the mitochondrial membrane potential, thus inhibiting further large-scale ROS production. Another important aspect is the chloroplast/cytosol/mitochondrion co-operation in green tissues under stress conditions aimed at modulating cell redox homeostasis. Durum wheat mitochondria may act against chloroplast/cytosol over-reduction: the malate/oxaloacetate antiporter and the rotenone-insensitive external NAD(P)H dehydrogenases allow cytosolic NAD(P)H oxidation; under stress this may occur without high ROS production due to co-operation with AOX, which is activated by intermediates of the photorespiratory cycle.

The transcript levels of two plant mitochondrial uncoupling protein (pUCP)-related genes are not affected by hyperosmotic stress in durum wheat seedlings showing an increased level of pUCP activity.

Etiolated early seedlings of durum wheat submitted to moderate and severe salt (NaCl) and osmotic (mannitol) stress showed no relevant increase of both transcript levels of two plant uncoupling protein (pUCP)-related genes and maximal pUCP activity in purified mitochondria (which estimates protein level); contrarily, pUCP functioning due to endogenous free fatty acids strongly increased. These results show that pUCP activation under hyperosmotic stress may be due to modulation of pUCP reaction rather than to an increased protein synthesis. Finally, a properly developed method, based on a single membrane potential measurement, to evaluate both pUCP maximal activity and functioning, is reported.

Seawater stress applied at germination affects mitochondrial function in durum wheat (Triticum durum) early seedlings.

Seawater stress effects on mitochondrial ATP synthesis and membrane potential (ΔΨ) were investigated in germinating durum wheat seedlings under moderate (22% seawater osmolarity, -0.62 MPa) and severe (37% seawater osmolarity, -1.04 MPa) stress. To estimate the osmotic component of salt stress, mannitol solutions (0.25 and 0.42 m) iso-osmotic with the saline ones were used. Moderate stress intensity only delayed mean germination time (MGT), whereas higher seawater osmolarity reduced germination percentage as well. In contrast, Na+ and Cl- accumulation showed a sharp increase under moderate stress and only a small further increase under severe stress, which was more pronounced for Cl-. Only severe stress significantly damaged succinate-dependent oxidative phosphorylation, which may be related to the stress-induced alteration in inner mitochondrial membrane permeability, as indicated by changes in ΔΨ profiles. Proline-dependent oxidative phosphorylation, however, was inhibited under moderate stress. This suggests the occurrence of an adaptation mechanism leading to proline accumulation as an osmoprotectant. Moreover, both the osmotic and the toxic components of seawater stress were detrimental to oxidative phosphorylation. Damage to germination and MGT, in contrast, were mainly caused by osmotic stress. Therefore, mitochondrial function appears to be a more sensitive target of toxic stress than growth. In conclusion, the effects of seawater stress on mitochondrial ATP synthesis vary in relation to the substrate oxidised and stress level, inducing both adaptive responses and damage.

A proteomic approach to study protein variation in GM durum wheat in relation to technological properties of semolina.

Genetic manipulation of durum wheats by tobacco rab-1 genes influence the trafficking of gluten proteins through the secretory system by up- or down-regulating the transport step from the ER to the Golgi apparatus which may in turn modify functional performance of the grain. Gluten proteins were extracted from two genetically manipulated lines - Svevo B730 1-1 and Ofanto B688 1-2 - and their control lines and were analyzed by two dimensional gel electrophoresis. When the two-dimensional maps were compared by image analysis no significant differences between the GM line with an up-regulated trafficking containing the wild type tobacco rab1 (Svevo B730 1-1) and its control (Svevo control). By contrast, significant differences were found between the GM line with a down-regulated trafficking due to the tobacco rab1 mutant form (Ofanto B688 1-2) and its control (Ofanto control). Of the new protein spots detected in the down-regulated Ofanto B688 1-2 map, only a beta-amylase was identified. The remaining spots were susceptible to chymotripsin action but not to trypsin one, as in the case of the gluten protein. Rheological measurements showed that gluten quality was enhanced in the down-regulated Ofanto B688 1-2 without an increase in the amount of gluten. Proteomics is a useful and powerful tool for investigating protein changes in GMOs and in understanding events in food science and technology.

Low temperature promotes intron retention in two e-cor genes of durum wheat.

Following the screening of a suppression subtractive library developed from durum wheat plants exposed to low temperature for 6 h, two early cold-regulated (e-cor) genes have been isolated. These genes, coding putatively for a ribokinase (7H8) and a C3H2C3 RING-finger protein (6G2), were characterized by the stress-induced retention of a subset of introns in the mature mRNA. This feature was dependent on cold for 7H8 and on cold and dehydration for 6G2. When other genes, such as the stress-related gene WCOR410c, coding for a dehydrin (one intron), or a gene coding for a putative ATP binding cassette transporter (16 introns) were analyzed, no cold-dependent intron retention was observed. Cold-induced intron retention was not observed in mutants defective in the chloroplast development; nevertheless treatment with cycloheximide in the absence of cold was able to promote intron retention for the 7H8 e-cor gene. These results suggest that the cold-induced intron retention reflects the response of the spliceosoma to specific environmental signals transduced to the splicing protein factors through a chloroplast-dependent pathway. Notably, when the 7H8 Arabidopsis orthologous gene was analyzed, no stress induction in terms of mRNA abundance and no cold-dependent intron retention was detected. Otherwise, 6G2 Arabidopsis homologous sequences sharing the same genomic structure of the durum wheat 6G2 showed a similar intron retention event although not strictly dependent on stress.

Chromosome regions and stress-related sequences involved in resistance to abiotic stress in Triticeae.

Drought, low temperature and salinity are the most important abiotic stress factors limiting crop productivity. A genomic map of major loci and QTLs affecting stress tolerance in Triticeae identified the crucial role of the group 5 chromosomes, where the highest concentration of QTLs and major loci controlling plant's adaptation to the environment (heading date, frost and salt tolerance) has been found. In addition, a conserved region with a major role in drought tolerance has been localized to the group 7 chromosomes. Extensive molecular biological studies have led to the cloning of many stress-related genes and responsive elements. The expression of some stress-related genes was shown to be linked to stress-tolerant QTLs, suggesting that these genes may represent the molecular basis of stress tolerance. The development of suitable genetic tools will allow the role of stress-related sequences and their relationship with stress-tolerant loci to be established in the near future.

Reactive oxygen species inhibit the succinate oxidation-supported generation of membrane potential in wheat mitochondria.

In order to gain a first insight into the effects of reactive oxygen species (ROS) on plant mitochondria, we studied the effect of the ROS producing system consisting of xanthine plus xanthine oxidase on the rate of membrane potential (DeltaPsi) generation due to either succinate or NADH addition to durum wheat mitochondria as monitored by safranin fluorescence. We show that the early ROS production inhibits the succinate-dependent, but not the NADH-dependent, DeltaPsi generation and oxygen uptake. This inhibition appears to depend on the impairment of mitochondrial permeability to succinate. It does not involve mitochondrial thiol groups sensitive to either mersalyl or N-ethylmaleimide and might involve both protein residues and/or membrane lipids, as suggested by the mixed nature. We propose that, during oxidative stress, early generation of ROS can affect plant mitochondria by impairing metabolite transport, thus preventing further substrate oxidation, DeltaPsi generation and consequent large-scale ROS production.