Genome-Wide Identification and Characterization of the Wheat Remorin (TaREM) Family during Cold Acclimation.

Remorins (REMs) are plant-specific proteins that play an essential role in plant-microbe interactions. However, their roles in vernalization and abiotic stress responses remain speculative. Most remorins have a variable proline-rich -half and a more conserved -half that is predicted to form coils. A search of the wheat ( L.) database revealed the existence of 20 different genes, which we classified into six groups on the basis of whether they shared a common phylogenetic and structural origin. Analysis of the physical genomic distributions demonstrated that genes are dispersed in the wheat genome and have one to seven introns. Promoter analysis of genes revealed the presence of putative -elements related to diverse functions like development, hormonal regulation, and biotic and abiotic stress responsiveness. Expression levels of genes were measured in plants grown under field and controlled conditions and in response to hormone treatment. Our analyses revealed that 12 members of the REM family are regulated during cold acclimation in wheat in four different tissues (roots, crowns, stems, and leaves), with the highest expression in roots. Differential gene expression was found between wheat cultivars with contrasting degrees of cold tolerance, suggesting the implication of genes in cold response and tolerance. Additionally, eight genes were induced in response to abscisic acid and methyl jasmonate treatment. This genome-wide analysis of genes provides valuable resources for functional analysis aimed at understanding their role in stress adaptation.

Transcriptomic Insights into Phenological Development and Cold Tolerance of Wheat Grown in the Field.

Cold acclimation and winter survival in cereal species is determined by complicated environmentally regulated gene expression. However, studies investigating these complex cold responses are mostly conducted in controlled environments that only consider the responses to single environmental variables. In this study, we have comprehensively profiled global transcriptional responses in crowns of field-grown spring and winter wheat (Triticum aestivum) genotypes and their near-isogenic lines with the VRN-A1 alleles swapped. This in-depth analysis revealed multiple signaling, interactive pathways that influence cold tolerance and phenological development to optimize plant growth and development in preparation for a wide range of over-winter stresses. Investigation of genetic differences at the VRN-A1 locus revealed that a vernalization requirement maintained a higher level of cold response pathways while VRN-A1 genetically promoted floral development. Our results also demonstrated the influence of genetic background on the expression of cold and flowering pathways. The link between delayed shoot apex development and the induction of cold tolerance was reflected by the gradual up-regulation of abscisic acid-dependent and C-REPEAT-BINDING FACTOR pathways. This was accompanied by the down-regulation of key genes involved in meristem development as the autumn progressed. The chromosome location of differentially expressed genes between the winter and spring wheat genetic backgrounds showed a striking pattern of biased gene expression on chromosomes 6A and 6D, indicating a transcriptional regulation at the genome level. This finding adds to the complexity of the genetic cascades and gene interactions that determine the evolutionary patterns of both phenological development and cold tolerance traits in wheat.

An integrative approach to identify hexaploid wheat miRNAome associated with development and tolerance to abiotic stress.

BACKGROUND: Wheat is a major staple crop with broad adaptability to a wide range of environmental conditions. This adaptability involves several stress and developmentally responsive genes, in which microRNAs (miRNAs) have emerged as important regulatory factors. However, the currently used approaches to identify miRNAs in this polyploid complex system focus on conserved and highly expressed miRNAs avoiding regularly those that are often lineage-specific, condition-specific, or appeared recently in evolution. In addition, many environmental and biological factors affecting miRNA expression were not yet considered, resulting still in an incomplete repertoire of wheat miRNAs. RESULTS: We developed a conservation-independent technique based on an integrative approach that combines machine learning, bioinformatic tools, biological insights of known miRNA expression profiles and universal criteria of plant miRNAs to identify miRNAs with more confidence. The developed pipeline can potentially identify novel wheat miRNAs that share features common to several species or that are species specific or clade specific. It allowed the discovery of 199 miRNA candidates associated with different abiotic stresses and development stages. We also highlight from the raw data 267 miRNAs conserved with 43 miRBase families. The predicted miRNAs are highly associated with abiotic stress responses, tolerance and development. GO enrichment analysis showed that they may play biological and physiological roles associated with cold, salt and aluminum (Al) through auxin signaling pathways, regulation of gene expression, ubiquitination, transport, carbohydrates, gibberellins, lipid, glutathione and secondary metabolism, photosynthesis, as well as floral transition and flowering. CONCLUSION: This approach provides a broad repertoire of hexaploid wheat miRNAs associated with abiotic stress responses, tolerance and development. These valuable resources of expressed wheat miRNAs will help in elucidating the regulatory mechanisms involved in freezing and Al responses and tolerance mechanisms as well as for development and flowering. In the long term, it may help in breeding stress tolerant plants.

Potential for increased photosynthetic performance and crop productivity in response to climate change: role of CBFs and gibberellic acid.

We propose that targeting the enhanced photosynthetic performance associated with the cold acclimation of winter cultivars of rye (Secale cereale L.), wheat (Triticum aestivum L.), and Brassica napus L. may provide a novel approach to improve crop productivity under abiotic as well as biotic stress conditions. In support of this hypothesis, we provide the physiological, biochemical, and molecular evidence that the dwarf phenotype induced by cold acclimation is coupled to significant enhancement in photosynthetic performance, resistance to photoinhibition, and a decreased dependence on photoprotection through non-photochemical quenching which result in enhanced biomass production and ultimately increased seed yield. These system-wide changes at the levels of phenotype, physiology, and biochemistry appear to be governed by the family of C-repeat/dehydration-responsive family of transcription factors (CBF/DREB1). We relate this phenomenon to the semi-dwarf, gibberellic acid insensitive (GAI), cereal varieties developed during the "green revolution" of the early 1960s and 1970s. We suggest that genetic manipulation of the family of C-repeat/dehydration-responsive element binding transcription factors (CBF/DREB1) may provide a novel approach for the maintenance and perhaps even the enhancement of plant productivity under conditions of sub-optimal growth conditions predicted for our future climate.

Transcriptome analysis of an mvp mutant reveals important changes in global gene expression and a role for methyl jasmonate in vernalization and flowering in wheat.

The einkorn wheat mutant mvp-1 (maintained vegetative phase 1) has a non-flowering phenotype caused by deletions including, but not limited to, the genes CYS, PHYC, and VRN1. However, the impact of these deletions on global gene expression is still unknown. Transcriptome analysis showed that these deletions caused the upregulation of several pathogenesis-related (PR) and jasmonate-responsive genes. These results suggest that jasmonates may be involved in flowering and vernalization in wheat. To test this hypothesis, jasmonic acid (JA) and methyl jasmonate (MeJA) content in mvp and wild-type plants was measured. The content of JA was comparable in all plants, whereas the content of MeJA was higher by more than 6-fold in mvp plants. The accumulation of MeJA was also observed in vernalization-sensitive hexaploid winter wheat during cold exposure. This accumulation declined rapidly once plants were deacclimated under floral-inductive growth conditions. This suggests that MeJA may have a role in floral transition. To confirm this result, we treated vernalization-insensitive spring wheat with MeJA. The treatment delayed flowering with significant downregulation of both TaVRN1 and TaFT1 genes. These data suggest a role for MeJA in modulating vernalization and flowering time in wheat.

Long-term growth under elevated CO2 suppresses biotic stress genes in non-acclimated, but not cold-acclimated winter wheat.

This study compared the photosynthetic performance and the global gene expression of the winter hardy wheat Triticum aestivum cv Norstar grown under non-acclimated (NA) or cold-acclimated (CA) conditions at either ambient CO2 or elevated CO2. CA Norstar maintained comparable light-saturated and CO2-saturated rates of photosynthesis but lower quantum requirements for PSII and non-photochemical quenching relative to NA plants even at elevated CO2. Neither NA nor CA plants were sensitive to feedback inhibition of photosynthesis at elevated CO2. Global gene expression using microarray combined with bioinformatics analysis revealed that genes affected by elevated CO2 were three times higher in NA (1,022 genes) compared with CA (372 genes) Norstar. The most striking effect was the down-regulation of genes involved in the plant defense responses in NA Norstar. In contrast, cold acclimation reversed this down-regulation due to the cold induction of genes involved in plant pathogenesis resistance; and cellular and chloroplast protection. These results suggest that elevated CO2 has less impact on plant performance and productivity in cold-adapted winter hardy plants in the northern climates compared with warmer environments. Selection for cereal cultivars with constitutively higher expression of biotic stress defense genes may be necessary under elevated CO2 during the warm growth period and in warmer climates.

Winter wheat hull (husk) is a valuable source for tricin, a potential selective cytotoxic agent.

The flavone, tricin (5,7,4'-trihydroxy-3',5'-dimethoxyflavone) has great potential as an anticancer agent, due to its specific chemopreventive activity. In spite of these characteristics, its use in preclinical studies is still limited, mainly because of its limited availability and high production cost. Tricin is found mainly in cereal grains, such as wheat, rice, barley, oat and maize. However, its concentration in these plants is not sufficient for commercial use. To find a reliable, rich source of tricin, we investigated its distribution in different parts of wheat (Triticum aestivum) and designed an efficient method for its isolation and purification. The highest amount (770 ± 157 mg/kg dry weight) was found in the husks of winter wheat. This concentration is one of the highest in any plant species and is considered as a cheap source of natural tricin. The purified wheat husks tricin was found to be a selective potent inhibitor of two cancer cell lines of liver and pancreas, while having no side effects on normal cells. This selective action suggests that tricin could be considered as a potential candidate for pre-clinical trials as a chemopreventive agent. In addition, fibre-rich crude wheat husk could be used as a natural chemopreventive agent in food supplement.

Tricin biosynthesis during growth of wheat under different abiotic stresses.

In plants, O-methylation is mediated by an enzyme family of O-methyltransferases (OMTs) that transfer the methyl groups from the methyl donor, S-adenosyl-L-methionine (AdoMet) to suitable phenolic acceptor molecules. In a previous study [1], a flavonoid OMT (TaOMT2) was isolated and characterized from wheat (Triticum aestivum L.) leaves. Its novel gene product catalyzes three sequential O-methylations of the flavone tricetin (5,7,3',4',5'-pentahydroxyflavone) to its 3'-monomethyl-(selgin)→3',5'-dimethyl-(tricin)→3',4',5'-trimethyl (TMT) ether derivatives, with tricin being the major product of the reaction. In this report, the biological significance of tricetin methylation was investigated by measuring the OMT activity, its expression level, and the accumulation of its major product (tricin) at different stages of development of wheat plants exposed to different abiotic stresses such as cold, salt and drought. The results showed that tricin accumulates mostly in wheat inflorescences under normal conditions compared to leaves and other developmental stages. Tricin accumulation was associated with increased TaOMT2 expression level and its enzyme activity, suggesting a possible de novo synthesis of the enzyme at this important developmental stage. This phenomenon may be attributed to the putative role of tricin in protecting seeds against biotic and abiotic stresses. The functions of tricin during growth and development of wheat and the importance of tricetin methylation during abiotic stresses are discussed.

Cryopreservation of insulin-secreting INS832/13 cells using a wheat protein formulation.

Diabetes is a global epidemic that affects about 285million people worldwide. For severely-ill patients with type I diabetes, whole pancreas or islet transplantation is the only therapeutic option. Islet transplantation is hindered by the scarce supply of fresh functional islets and limitations in cryopreservation procedures. Thus, improved cryopreservation procedures are needed to increase the availability of functional islets for clinical applications. Towards this goal, this work developed a cryopreservation protocol for pancreatic cells using proteins that accumulate naturally in freezing-tolerant plants. A preincubation of cells with 1% lecithin-1% glycerol-1% N-methylpyrrolidone followed by cryopreservation with partially purified proteins from wheat improved the viability and insulin-secreting properties of INS832/13 cells, compared to cryopreservation with 10% dimethyl sulfoxide (Me2SO). The major factor that enhanced the cryoprotective effect of the wheat protein formulation was preincubation with the lipid lecithin. Expression profiles of genes involved in metabolic and signaling functions of pancreatic cells (Ins, Glut1/2/3, Pdx1, Reg1α) were similar between fresh cells and those cryopreserved with the plant protein formulation. This novel plant-based technology, which is non-toxic and contains no animal material, is a promising alternative to Me2SO for cryopreservation of insulin-secreting pancreatic cells.

Daphnetin methylation stabilizes the activity of phosphoribulokinase in wheat during cold acclimation.

The methylation of daphnetin (7,8-dihydroxycoumarin) to its 8-methyl derivative is catalyzed by a wheat (Triticum aestivum L.) O-methyltransferase (TaOMT1). This enzyme is regulated by cold and photosystem II excitation pressure (plastid redox state). Here, we investigated the biological significance of this methylation and its potential role in modulating the activity of kinases in wheat. To identify the potential kinases that may interact with daphnetin in wheat, the soluble protein extract from aerial parts of cold-acclimated wheat was purified by DEAE-cellulose separation and affinity chromatography on a daphnetin derivative (7,8-dihydroxy-4-coumarin acetic acid)-EAH sepharose column. Mass spectrometric analysis indicated that wheat phosphoribulokinase (TaPRK) is the major kinase that binds to daphnetin. This TaPRK plays an important role in regulating the flow of carbon through the Calvin cycle, by catalyzing the final step in the regeneration of ribulose 1,5-bisphosphate from ribulose-5-phosphate (Ru5P) and ATP. The activities of TaPRK, endogenous or recombinant, are inhibited by daphnetin in a specific and dose-dependent manner, but not by its monomethyl derivative (7-methyl, 8-hydroxycoumarin). Furthermore, HPLC-MS analysis of wheat extracts reveals that 7,8-dimethoxycoumarin is more abundant than its monomethyl derivative. The results also show that cold acclimation does not alter the level of TaPRK mRNA or its enzyme activity, and thus ensures the stable generation of ribulose 1,5-biphosphate.

Expression of vernalization responsive genes in wheat is associated with histone H3 trimethylation.

The transition to flowering in winter wheat requires prolonged exposure to low temperature, a process called vernalization. This process is regulated by a genetic pathway that involves at least three genes, Triticum aestivum VERNALIZATION 1 (TaVRN1), Triticum aestivum VERNALIZATION 2 (TaVRN2) and Triticum aestivum FLOWERING LOCUS T-like 1 (TaFT1). These genes regulate flowering by integrating environmental and developmental cues. To determine whether the expression of these genes is associated with the chromatin methylation state during vernalization in wheat, the level of two markers of histone modifications, the activator histone H3 trimethylation of lysine 4 (H3K4me3) and the repressor histone H3 trimethylation of lysine 27 (H3K27me3) were measured at the promoter regions of these three genes. Bioinformatics analysis of these promoters demonstrates the presence of conserved cis-acting elements in the promoters of the three vernalization genes, TaVRN1, TaVRN2 and TaFT1. These elements are targeted by common transcription factors in the vernalization responsive cereals. These promoters also contain the functional "units" PRE/TRE targeted by Polycomb and Trithorax proteins that maintain repressed or active transcription states of developmentally regulated genes. These proteins are known to be associated with the regulation of H3K4me3 and H3K27me3. Expression studies indicate that TaVRN1 and TaFT1 are up-regulated by vernalization in winter wheat. This up-regulation is associated with increased level of the activator H3K4me3 with no change in the level of the repressor H3K27me3 at the promoter region. This study shows that the flowering transition induced by vernalization in winter wheat is associated with histone methylation at the promoter level of TaVRN1 and TaFT1 while the role of these markers is less evident in TaVRN2 repression. This may represent part of the cellular memory of vernalization in wheat.

The effects of phenotypic plasticity on photosynthetic performance in winter rye, winter wheat and Brassica napus.

The contributions of phenotypic plasticity to photosynthetic performance in winter (cv Musketeer, cv Norstar) and spring (cv SR4A, cv Katepwa) rye (Secale cereale) and wheat (Triticum aestivum) cultivars grown at either 20°C [non-acclimated (NA)] or 5°C [cold acclimated (CA)] were assessed. The 22-40% increase in light-saturated rates of CO2 assimilation in CA vs NA winter cereals were accounted for by phenotypic plasticity as indicated by the dwarf phenotype and increased specific leaf weight. However, phenotypic plasticity could not account for (1) the differential temperature sensitivity of CO2 assimilation and photosynthetic electron transport, (2) the increased efficiency and light-saturated rates of photosynthetic electron transport or (3) the decreased light sensitivity of excitation pressure and non-photochemical quenching between NA and NA winter cultivars. Cold acclimation decreased photosynthetic performance of spring relative to winter cultivars. However, the differences in photosynthetic performances between CA winter and spring cultivars were dependent upon the basis on which photosynthetic performance was expressed. Overexpression of BNCBF17 in Brassica napus generally decreased the low temperature sensitivity (Q10) of CO2 assimilation and photosynthetic electron transport even though the latter had not been exposed to low temperature. Photosynthetic performance in wild type compared to the BNCBF17-overexpressing transgenic B. napus indicated that CBFs/DREBs regulate not only freezing tolerance but also govern plant architecture, leaf anatomy and photosynthetic performance. The apparent positive and negative effects of cold acclimation on photosynthetic performance are discussed in terms of the apparent costs and benefits of phenotypic plasticity, winter survival and reproductive fitness.

Changes in wheat leaf phenolome in response to cold acclimation.

A study of wheat (Triticum aestivum L.) leaves phenolome was carried out during cold acclimation of the winter (Claire) and spring (Bounty) varieties using a combination of HPLC-ESI-MS techniques. A total of 40 phenolic and flavonoid compounds were identified, and consisted mainly of two coumarin derivatives, eight simple phenolic derivatives, 10 hydroxycinnamoyl amides and 20 flavonoid derivatives. Identification and quantification of individual compounds were performed using an HPLC system coupled with a photodiode array detector and two different ESI-MS systems, in combination with a multiple reaction monitoring (MRM) technique. The analyses indicated that, although there were no qualitative differences in their profiles, the winter variety exhibited a higher phenolic content compared to the spring variety when both were grown under non-acclimated (control) conditions. Cold acclimation, on the other hand, resulted in a significant differential accumulation of phenolic compounds in both varieties: mostly as luteolin C-glycosides and their O-methyl derivatives in the winter variety (Claire) and a derivative of hydroxycinnamoyl amide in the spring variety (Bounty). These compounds accumulated in relatively large amounts in the apoplastic compartment. The accumulation of the O-methylated derivatives was associated with a marked increase in O-methyltransferase (OMT) activity. In addition, the trimethylated flavone, 3',4',5'-trimethyltricetin was identified for the first time in the native extracts of both control and cold-acclimated wheat leaves. The accumulation of a mixture of beneficial flavonoids, such as iso-orientin, vitexin and tricin in cold acclimated wheat leaves, attests for its potential as an inexpensive source of a health-promoting supplement to the human diet.

Flagellin produced in plants is a potent adjuvant for oral immunization.

The aim of this study was to produce adjuvant with high biosafety, efficacy and low cost. Towards this goal, the plant Nicotiana benthamiana transient expression system was successfully used to express Salmonella typhimurium's flagellin (FljB). The yield of the expressed FljB was 280 mg per kg of fresh weight (FW) leaves. The lyophilized plant powder containing plant expressing FljB was mixed with ovalbumin (OVA) and used for oral immunization of BALB/c mice. The ELISA analysis showed higher and accelerated OVA-specific serum antibody responses in mice given the mixture when compared to animals receiving OVA alone. Furthermore, FljB elicited a mixed Th1/Th2 response as shown by the presence of specific anti-OVA IgG1, IgG2a and IgG2b isotypes. OVA-specific IgAs were also detected in mice given the mixture. Cell-mediated immune response to OVA was induced by FljB as determined by a spleen lymphocyte specific proliferation test. No immune response was generated against FljB. In conclusion, our results showed for the first time the production of FljB in plants and the efficient use of the crude lyophilized extract as an adjuvant for oral immunization.

Production of human rotavirus and Salmonella antigens in plants and elicitation of fljB-specific humoral responses in mice.

A Nicotiana benthamiana transient expression system was used to express single antigen and dimeric combinations of the human rotavirus (HRV) VP7 and a truncated VP4 (VP4Δ) proteins fused with Salmonella typhimurium's flagellin fljB subunit. Immunoblot analyses using rabbit antibodies generated against these proteins demonstrated that the constructs were successfully expressed with yields ranging from 0.85 to 31.97 µg of recombinant protein per gram of fresh leaf tissue. Expressing the single and dimeric antigens has no effect on plant growth and development except for VP7 and VP4Δ::VP7, which show mild necrotic lesions. Immunization of mice with proteins from leaves transformed with constructs bearing the fljB moiety elicited an fljB-specific humoral response. The Nicotiana benthamiana transient system is efficient to express multiple combinations of pathogen proteins and demonstrates the potential of generating a Salmonella typhimurium subunit vaccine in plants.

Carboxymethyl-starch excipients for gastrointestinal stable oral protein formulations containing protease inhibitors.

PURPOSE: The aim of this study was to develop a formulation for bioactive compounds using Carboxymethyl Starch (CMS) as excipient containing protease inhibitors. This formulation provided gastro protection and enhanced stability against pancreatic enzymes. Such stability is needed for formulation of oral vaccines with specific antigens. METHODS: CMS was synthesized by treatment of starch with monochloroacetic acid in conditions leading to a substitution degree of about 1 meq/g and used as excipient for monolithic devices (300 mg tablets). Pefabloc SC and Aprotinin inhibitors were tested in dissolution media and in formulation to prevent the degradation of released bioactive materials. To evaluate the structural integrity and biological stability of plant proteins in the CMS formulation, albumin and lipase were added to the plant protein extract as protein and respectively as enzyme markers. The amounts of released and recovered proteins were evaluated by SDS-PAGE and densitometric analysis. RESULTS: It was found that 1.6 % (w/w) of Pefabloc SC provides 98 % protection of the released plant proteins for formulations of 30 % alfalfa protein extract (APE) with CMS. In addition, when bovine serum albumin (BSA) added to the plant protein extract as a marker, 90 % protection of the released BSA was observed. Furthermore, a much higher lipase activity was found in the releasing media when the formulations contained Pefabloc SC. CONCLUSION: Formulations with CM-Starch excipients and containing protease inhibitors prevent protein degradation and protect lipase activity, showing a marked potential to use for orally administered bioactive peptides and therapeutic enzymes.

Heterologous expression of wheat VERNALIZATION 2 (TaVRN2) gene in Arabidopsis delays flowering and enhances freezing tolerance.

The vernalization gene 2 (VRN2), is a major flowering repressor in temperate cereals that is regulated by low temperature and photoperiod. Here we show that the gene from Triticum aestivum (TaVRN2) is also regulated by salt, heat shock, dehydration, wounding and abscissic acid. Promoter analysis indicates that TaVRN2 regulatory region possesses all the specific responsive elements to these stresses. This suggests pleiotropic effects of TaVRN2 in wheat development and adaptability to the environment. To test if TaVRN2 can act as a flowering repressor in species different from the temperate cereals, the gene was ectopically expressed in the model plant Arabidopsis. Transgenic plants showed no alteration in morphology, but their flowering time was significantly delayed compared to controls plants, indicating that TaVRN2, although having no ortholog in Brassicaceae, can act as a flowering repressor in these species. To identify the possible mechanism by which TaVRN2 gene delays flowering in Arabidopsis, the expression level of several genes involved in flowering time regulation was determined. The analysis indicates that the late flowering of the 35S::TaVRN2 plants was associated with a complex pattern of expression of the major flowering control genes, FCA, FLC, FT, FVE and SOC1. This suggests that heterologous expression of TaVRN2 in Arabidopsis can delay flowering by modulating several floral inductive pathways. Furthermore, transgenic plants showed higher freezing tolerance, likely due to the accumulation of CBF2, CBF3 and the COR genes. Overall, our data suggests that TaVRN2 gene could modulate a common regulator of the two interacting pathways that regulate flowering time and the induction of cold tolerance. The results also demonstrate that TaVRN2 could be used to manipulate flowering time and improve cold tolerance in other species.

Wheat proteins enhance stability and function of adhesion molecules in cryopreserved hepatocytes.

Cryopreserved hepatocytes with good hepatospecific functions upon thawing are important for clinical transplantation and for in vitro drug toxicity testing. However, cryopreservation reduces viability and certain hepatospecific functions, but the most pronounced change is diminished attachment efficiency of hepatocytes. Adhesion of cells to the extracellular matrix and cell-cell contacts are crucial for many aspects of cellular function. These processes are partly mediated and controlled by cellular adhesion molecules. The mechanisms responsible for reduced attachment efficiency of cryopreserved hepatocytes are not well understood. To address this question, we investigated the effect of a new cryopreservation procedure, using wheat proteins (WPs) or mixtures of recombinant forms of wheat freezing tolerance-associated proteins, on the stability of three important adhesion molecules (beta1-integrin, E-cadherin, and beta-catenin). Immunoblot analyses revealed that the levels of beta1-integrin, E-cadherin, and beta-catenin were much lower in cryopreserved rat hepatocytes, when compared to fresh cells. Protein expression of the adhesion molecules was generally lower in cells cryopreserved with DMSO, compared to WPs. Moreover, the stability of the adhesion molecules was not affected by cryopreservation to the same degree, with more pronounced decreases occurring for beta1-integrin (62-74%) > beta-catenin (51-58%) > E-cadherin (21-37%). However, when hepatocytes were cryopreserved with partially purified WPs (SulWPE, AcWPE) or with mixtures of recombinant wheat proteins, there was a clear protective effect against the loss of protein expression of beta1-integrin, E-cadherin, and beta-catenin. Protein expression was only 10-20% lower than that observed in fresh hepatocytes. These findings clearly demonstrate that WPs, and more particularly, partially purified WPs and recombinant wheat proteins, were more efficient for cryopreservation of rat hepatocytes by maintaining good expression of these adhesion molecules. These promising results could lead to a new and improved cryopreservation technology for applications such as clinical transplantation of hepatocytes.

Wheat proteins improve cryopreservation of rat hepatocytes.

Hepatocytes are an important physiological model for in vitro studies of drug metabolism and toxicity. However, fresh hepatocytes are not always available and hence cyopreservation is needed to preserve large quantities until they are needed for these applications. Hepatocytes are extremely sensitive to damage induced by the freeze-thaw process, even after addition of traditional cryoprotectants such as dimethyl sulfoxide (DMSO). Furthermore, they do not proliferate in culture. We previously demonstrated that a crude wheat extract protects rat hepatocytes during cryopreservation and could provide a promising alternative to DMSO. We have considerably improved this novel cryopreservation procedure by using wheat extracts that are partially purified by either ammonium sulphate or acetone precipitation, or by using recombinant wheat freezing tolerance-associated proteins such as WCS120, TaTIL, WCS19, and TaIRI-2. These improved procedures enhance long-term storage (2-12 months) and recovery of large quantities of healthy cells after cryopreservation, and maintain the differentiated functions of rat hepatocytes, compared to freshly isolated cells, as judged by viability (77-93%), adherence (77%) and metabolic functions of major cytochrome P450 isoforms CYP1A1/2, CYP2C6, CYP2D2, and CYP3A1/2. The advantage of using wheat proteins as cryopreservants is that they are non-toxic, natural products that do not require animal serum, and are economical and easy to prepare.