Modulation of the wheat transcriptome by TaZFP13D under well-watered and drought conditions.

Maintaining global food security in the context of climate changes will be an important challenge in the next century. Improving abiotic stress tolerance of major crops such as wheat can contribute to this goal. This can be achieved by the identification of the genes involved and their use to develop tools for breeding programs aiming to generate better adapted cultivars. Recently, we identified the wheat TaZFP13D gene encoding Zinc Finger Protein 13D as a new gene improving water-stress tolerance. The current work analyzes the TaZFP13D-dependent transcriptome modifications that occur in well-watered and dehydration conditions to better understand its function during normal growth and during drought. Plants that overexpress TaZFP13D have a higher biomass under well-watered conditions, indicating a positive effect of the protein on growth. Survival rate and stress recovery after a severe drought stress are improved compared to wild-type plants. The latter is likely due the higher activity of key antioxidant enzymes and concomitant reduction of drought-induced oxidative damage. Conversely, down-regulation of TaZFP13D decreases drought tolerance and protection against drought-induced oxidative damage. RNA-Seq transcriptome analysis identified many genes regulated by TaZFP13D that are known to improve drought tolerance. The analysis also revealed several genes involved in the photosynthetic electron transfer chain known to improve photosynthetic efficiency and chloroplast protection against drought-induced ROS damage. This study highlights the important role of TaZFP13D in wheat drought tolerance, contributes to unravel the complex regulation governed by TaZFPs, and suggests that it could be a promising marker to select wheat cultivars with higher drought tolerance.

Control of stress-induced apoptosis by freezing tolerance-associated wheat proteins during cryopreservation of rat hepatocytes.

Cryopreservation is used for long-term storage of cells and tissues. Cryoprotectants such as dimethyl disulfoxide (DMSO) are used to protect cells against freeze-thaw damage. Despite the use of cryoprotectants, hepatocytes are sensitive to stresses imposed by freeze and thaw processes, which cause physical damage, loss of functionality, or cell death. As an alternative, we have developed new technology using several recombinant wheat proteins as cryoprotectants: TaENO (enolase), TaBAS1 (2-Cys peroxiredoxin), and a combination of WCS120 (dehydrin) with TaIRI-2 (inhibitor of ice recrystallization). This study aims to understand the mechanisms by which these plant proteins protect rat hepatocytes against cell death incurred during cryopreservation. Our analysis revealed that for cells cryopreserved with DMSO, cell death occurred by apoptosis and necrosis. Apoptosis was detected by activation of effector caspases-3 and -7, PARP cleavage, and nuclear chromatin condensation. These apoptotic events were inhibited when hepatocytes were cryopreserved with the different plant proteins. Cryopreservation with DMSO activated apoptosis through the mitochondrial pathway: the Bax/Bcl-2 protein ratio increased, mitochondrial membrane potential decreased, and initiator caspase-9 was activated. Furthermore, the endoplasmic reticulum pathway of apoptosis was activated: levels of the chaperone Bip/GRP78 decreased, pro-apoptotic transcription factor CHOP was induced, and initiator caspase-12 was activated. Activation of the mitochondrial and endoplasmic reticulum pathways of apoptosis was attenuated when hepatocytes were cryopreserved with the different recombinant proteins. This study improves understanding of mechanisms of cryoprotection provided by these plant proteins during freezing stress. These proteins are natural products and show promising potential by decreasing cell death during cryopreservation of hepatocytes.

The barley stripe mosaic virus expression system reveals the wheat C2H2 zinc finger protein TaZFP1B as a key regulator of drought tolerance.

BACKGROUND: Drought stress is one of the major factors limiting wheat production globally. Improving drought tolerance is important for agriculture sustainability. Although various morphological, physiological and biochemical responses associated with drought tolerance have been documented, the molecular mechanisms and regulatory genes that are needed to improve drought tolerance in crops require further investigation. We have used a novel 4-component version (for overexpression) and a 3-component version (for underexpression) of a barley stripe mosaic virus-based (BSMV) system for functional characterization of the C2H2-type zinc finger protein TaZFP1B in wheat. These expression systems avoid the need to produce transgenic plant lines and greatly speed up functional gene characterization. RESULTS: We show that overexpression of TaZFP1B stimulates plant growth and up-regulates different oxidative stress-responsive genes under well-watered conditions. Plants that overexpress TaZFP1B are more drought tolerant at critical periods of the plant's life cycle. Furthermore, RNA-Seq analysis revealed that plants overexpressing TaZFP1B reprogram their transcriptome, resulting in physiological and physical modifications that help wheat to grow and survive under drought stress. In contrast, plants transformed to underexpress TaZFP1B are significantly less tolerant to drought and growth is negatively affected. CONCLUSIONS: This study clearly shows that the two versions of the BSMV system can be used for fast and efficient functional characterization of genes in crops. The extent of transcriptome reprogramming in plants that overexpress TaZFP1B indicates that the encoded transcription factor is a key regulator of drought tolerance in wheat.

Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells.

Efficient cryopreservation of cells at ultralow temperatures requires the use of substances that help maintain viability and metabolic functions post-thaw. We are developing new technology where plant proteins are used to substitute the commonly-used, but relatively toxic chemical dimethyl sulfoxide. Recombinant forms of four structurally diverse wheat proteins, TaIRI-2 (ice recrystallization inhibition), TaBAS1 (2-Cys peroxiredoxin), WCS120 (dehydrin), and TaENO (enolase) can efficiently cryopreserve hepatocytes and insulin-secreting INS832/13 cells. This study shows that TaIRI-2 and TaENO are internalized during the freeze-thaw process, while TaBAS1 and WCS120 remain at the extracellular level. Possible antifreeze activity of the four proteins was assessed. The "splat cooling" method for quantifying ice recrystallization inhibition activity (a property that characterizes antifreeze proteins) revealed that TaIRI-2 and TaENO are more potent than TaBAS1 and WCS120. Because of their ability to inhibit ice recrystallization, the wheat recombinant proteins TaIRI-2 and TaENO are promising candidates and could prove useful to improve cryopreservation protocols for hepatocytes and insulin-secreting cells, and possibly other cell types. TaENO does not have typical ice-binding domains, and the TargetFreeze tool did not predict an antifreeze capacity, suggesting the existence of nontypical antifreeze domains. The fact that TaBAS1 is an efficient cryoprotectant but does not show antifreeze activity indicates a different mechanism of action. The cryoprotective properties conferred by WCS120 depend on biochemical properties that remain to be determined. Overall, our results show that the proteins' efficiencies vary between cell types, and confirm that a combination of different protection mechanisms is needed to successfully cryopreserve mammalian cells.

Plant protein 2-Cys peroxiredoxin TaBAS1 alleviates oxidative and nitrosative stresses incurred during cryopreservation of mammalian cells.

There is increasing demand for cryopreserved cells such as liver and pancreatic cells for clinical applications. Cryopreservation at ultra-low temperatures requires use of cryoprotectants (e.g., dimethyl sulfoxide (DMSO)) to maintain cell integrity during freezing and thawing processes. Standard cryoprotectants are cytotoxic and more effective cryopreservation technologies are urgently needed for long-term storage of cells. As an alternative, soluble protein extracts (WPE) from winter wheat successfully replaced DMSO as a cryoprotectant for several mammalian cell types. To identify novel cryoactive proteins, the WPE was separated by chromatography and cryoactive fractions were analyzed by mass spectrometry. The wheat protein 2-Cys peroxiredoxin BAS1 (renamed TaBAS1) was identified as a potential cryoactive candidate. Recombinant proteins were prepared and found to possess dual functions as a peroxidase antioxidant and molecular chaperone, and display cryoprotective properties for hepatocytes and insulin-secreting INS832/13 cells. Following cryopreservation with TaBAS1, cells were plateable and showed high post-thaw viability, good adhesion properties, and well-maintained cell-specific metabolic functions. The overall quality of these cell types was equivalent or improved compared to cells that were cryopreserved with DMSO. The antioxidant and chaperone functions of TaBAS1 likely explain its efficacy in reducing oxidative/nitrosative stresses in cryopreserved cells. The plant protein TaBAS1 could be a promising molecule to include in cryostorage protocols. Biotechnol. Bioeng. 2016;113: 1511-1521. © 2015 Wiley Periodicals, Inc.

Wheat enolase demonstrates potential as a non-toxic cryopreservation agent for liver and pancreatic cells.

Cryopreservation is essential for long-term storage of cells and tissues, which can be used for clinical applications such as drug toxicity testing, human transplantation, reproductive, regenerative and transfusion medicine. It requires use of cryoprotectants (e.g. dimethyl disulfoxide (DMSO), glycerol) that protect cells and tissues from dehydration and damage caused by formation of intracellular ice during freezing. As an alternative to these cytotoxic cryoprotectants, we are developing new technology using natural substances produced by plants that survive freezing conditions. We previously showed that soluble protein extracts such as wheat protein extract (WPE) prepared from winter wheat plants can substitute for DMSO as a cryoprotectant for certain mammalian cell types. To identify novel cryoactive proteins, WPE was separated using different chromatographic procedures and cryoactive fractions were analyzed by mass spectrometry. The analysis revealed enolase as a potential wheat protein candidate. A recombinant enolase protein was prepared and was able to successfully cryopreserve rat hepatocytes and insulin-secreting INS832/13 pancreatic cells. Post-thaw cells had high viability and levels of metabolic activities. Cryopreserved cells were plateable and had good adherence and morphological properties. These results indicate that individual plant proteins such as enolase have promising potential as new, non-toxic technology for cryopreservation protocols used for clinical applications.

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.

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.

The chloroplastic lipocalin AtCHL prevents lipid peroxidation and protects Arabidopsis against oxidative stress.

Lipocalins are small ligand-binding proteins with a simple tertiary structure that gives them the ability to bind small, generally hydrophobic, molecules. Recent studies have shown that animal lipocalins play important roles in the regulation of developmental processes and are involved in tolerance to oxidative stress. Plants also possess various types of lipocalins, and bioinformatics analyses have predicted that some lipocalin members may be present in the chloroplast. Here we report the functional characterization of the Arabidopsis thaliana chloroplastic lipocalin AtCHL. Cellular fractionation showed that AtCHL is a thylakoid lumenal protein. Drought, high light, paraquat and abscisic acid treatments induce AtCHL transcript and protein accumulation. Under normal growth conditions, knockout (KO) and over-expressing (OEX) lines do not differ from wild-type plants in terms of phenotype and photosynthetic performance. However, KO plants, which do not accumulate AtCHL, show more damage upon photo-oxidative stress induced by drought, high light or paraquat. In contrast, a high level of AtCHL allows OEX plants to cope better with these stress conditions. When exposed to excess light, KO plants display a rapid accumulation of hydroxy fatty acids relative to the wild-type, whereas the lipid peroxidation level remains very low in OEX plants. The increased lipid peroxidation in KO plants is mediated by singlet oxygen and is not correlated with photo-inhibition of the photosystems. This work provides evidence suggesting that AtCHL is involved in the protection of thylakoidal membrane lipids against reactive oxygen species, especially singlet oxygen, produced in excess light.

Effects of government policies on the work of home care personnel and their occupational health and safety.

The health sector in Québec (Canada) is dealing with profound macro-economic and macro-organizational changes. This article is interested in the impact of these changes on the work of home health aides (HHAs) and home care nurses and their occupational health and safety (OHS). The study was carried out in the home care services (HCS) of four local community service centres (CLSC) with different organizational characteristics. It is based on an analysis by triangulation of 66 individual and group interviews, 22 observed workdays and 35 observed multidisciplinary or professional meetings, as well as on administrative documents. HHAs are experiencing an erosion of their job because the relational and affective aspects of their work are disappearing. This may be due to an increase in their physical workload, leading to an increase in musculoskeletal problems and, to a lesser extent, in psychological health problems. Nurses are seeing an increase in the volume of invisible work that they have to do, which also has the effect of decreasing the relational aspects of their activity. The increasingly numerous psychological health problems are the consequence of this change in their profession. This study also shows that managers' decisions at the local level can reduce or increase the work constraints of HHAs and nurses. Examples of good practices for HHAs are the stabilization of clienteles and the possibility of organizing their itinerary, while for nurses, it is in how clientele follow-up tools are implemented. This article discusses the effects of government policies and decisions on the work and OHS of home care personnel. To address this subject, we use a specific analysis of the workload of home health aides (HHAs) and nurses. We will show the relationships between managers' organizational choices to respond to governmental constraints and the resulting work changes. We will also look at their consequences on occupational health and safety (OHS) and on the work of different personnel.

The plant Apolipoprotein D ortholog protects Arabidopsis against oxidative stress.

BACKGROUND: Lipocalins are a large and diverse family of small, mostly extracellular proteins implicated in many important functions. This family has been studied in bacteria, invertebrate and vertebrate animals but little is known about these proteins in plants. We recently reported the identification and molecular characterization of the first true lipocalins from plants, including the Apolipoprotein D ortholog AtTIL identified in the plant model Arabidopsis thaliana. This study aimed to determine its physiological role in planta. RESULTS: Our results demonstrate that the AtTIL lipocalin is involved in modulating tolerance to oxidative stress. AtTIL knock-out plants are very sensitive to sudden drops in temperature and paraquat treatment, and dark-grown plants die shortly after transfer to light. These plants accumulate a high level of hydrogen peroxide and other ROS, which causes an oxidative stress that is associated with a reduction in hypocotyl growth and sensitivity to light. Complementation of the knock-out plants with the AtTIL cDNA restores the normal phenotype. On the other hand, overexpression enhances tolerance to stress caused by freezing, paraquat and light. Moreover, this overexpression delays flowering and maintains leaf greenness. Microarray analyses identified several differentially-regulated genes encoding components of oxidative stress and energy balance. CONCLUSION: This study provides the first functional evidence that a plant lipocalin is involved in modulating tolerance to oxidative stress. These findings are in agreement with recently published data showing that overexpression of ApoD enhances tolerance to oxidative stress and increases life span in mice and Drosophila. Together, the three papers strongly support a similar function of lipocalins in these evolutionary-distant species.

Overexpression of TaVRN1 in Arabidopsis promotes early flowering and alters development.

TaVRN1, a member of the APETALA1 (AP1) subfamily of MADS-box transcription factors, is a key gene that controls transition from vegetative to reproductive phase in wheat. The accumulation of TaVRN1 transcripts in winter wheat probably requires the down-regulation of TaVRT2, a MADS-box factor that binds and represses the TaVRN1 promoter, and of the flowering repressor TaVRN2. However, the molecular mechanisms by which TaVRN1 functions as an activator of phase transition is unknown. To address this, a combination of gene expression and functional studies was used. RNA in situ hybridization studies showed that TaVRN1 transcripts accumulate in all meristems and primordia associated with flower development. An interaction screen in yeast revealed that TaVRN1 interacts with several proteins involved in different processes of plant development such as transcription factors, kinases and a cyclophilin. Arabidopsis plants overexpressing TaVRN1 flower early and show various levels of modified plant architecture. The ectopic expression causes an overexpression of the AP1 and MAX4 genes, which are associated with flowering and auxin regulation, respectively. The induction of gene expression probably results from the binding of TaVRN1 to CArG motifs present on the AP1 and MAX4 promoters. In contrast, Arabidopsis plants that overexpress TaVRT2, which encodes a putative flowering repressor, show an opposite late flowering phenotype. Together, the data provide molecular evidence that TaVRN1 may have pleiotropic effects in various processes such as control of axillary bud growth, transition to flowering and development of floral organs.

TaVRT2 represses transcription of the wheat vernalization gene TaVRN1.

In wheat, VRN1/TaVRN1 and VRN2/TaVRN2 determine the growth habit and flowering time. In addition, the MADS box transcription factor VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (TaVRT2) is also associated with the vernalization response in a manner similar to TaVRN2. However, the molecular relationship between these three genes and their products is unknown. Using transient expression assays in Nicotiana benthamiana, we show that TaVRT2 acts as a repressor of TaVRN1 transcription. TaVRT2 binds the CArG motif in the TaVRN1 promoter and represses its activity in vivo. In contrast, TaVRN2 does not bind the TaVRN1 promoter and has no direct effect on its activity, but it can enhance the repression effect of TaVRT2. This suggests that a repressor complex regulates the expression of TaVRN1. In winter wheat, TaVRT2, TaVRN2 and TaVRN1 transcripts accumulate in the shoot apical meristem and young leaves, and temporal expression is consistent with TaVRT2 and TaVRN2 being repressors of floral transition, whereas TaVRN1 is an activator. Non-vernalized spring wheat grown under a short-day photoperiod accumulates TaVRT2 and shows a delay in flowering, suggesting that TaVRT2 is regulated independently by photoperiod and low temperature. The data presented suggest that TaVRT2, in association with TaVRN2, represses the transcription of TaVRN1.

Restructuring of the Québec health network and its effects on the profession of home health aides and their occupational health and safety.

The Québec health sector is facing profound macroeconomic and macro-organizational changes. This article addresses the impact of these changes on the work of home health aides (HHAs) and their occupational health and safety (OHS). The study was carried out in the home care services of four local community service centers (CLSCs) with different organizational characteristics. It is based on an analysis by triangulation of 66 individual and group interviews, 11 work days, and 35 multidisciplinary or professional meetings observed, as well as administrative documents. HHAs are experiencing an erosion of their job because the relational and emotional components of their work are disappearing. This results in an increase in musculoskeletal and psychological health problems. This study also shows that managers' decisions can reduce or increase the HHAs' work constraints. Stability in the clientele served and the possibility of organizing their routes are good examples of positive impacts.

Wheat EST resources for functional genomics of abiotic stress.

BACKGROUND: Wheat is an excellent species to study freezing tolerance and other abiotic stresses. However, the sequence of the wheat genome has not been completely characterized due to its complexity and large size. To circumvent this obstacle and identify genes involved in cold acclimation and associated stresses, a large scale EST sequencing approach was undertaken by the Functional Genomics of Abiotic Stress (FGAS) project. RESULTS: We generated 73,521 quality-filtered ESTs from eleven cDNA libraries constructed from wheat plants exposed to various abiotic stresses and at different developmental stages. In addition, 196,041 ESTs for which tracefiles were available from the National Science Foundation wheat EST sequencing program and DuPont were also quality-filtered and used in the analysis. Clustering of the combined ESTs with d2_cluster and TGICL yielded a few large clusters containing several thousand ESTs that were refractory to routine clustering techniques. To resolve this problem, the sequence proximity and "bridges" were identified by an e-value distance graph to manually break clusters into smaller groups. Assembly of the resolved ESTs generated a 75,488 unique sequence set (31,580 contigs and 43,908 singletons/singlets). Digital expression analyses indicated that the FGAS dataset is enriched in stress-regulated genes compared to the other public datasets. Over 43% of the unique sequence set was annotated and classified into functional categories according to Gene Ontology. CONCLUSION: We have annotated 29,556 different sequences, an almost 5-fold increase in annotated sequences compared to the available wheat public databases. Digital expression analysis combined with gene annotation helped in the identification of several pathways associated with abiotic stress. The genomic resources and knowledge developed by this project will contribute to a better understanding of the different mechanisms that govern stress tolerance in wheat and other cereals.

Identification, expression, and evolutionary analyses of plant lipocalins.

Lipocalins are a group of proteins that have been characterized in bacteria, invertebrate, and vertebrate animals. However, very little is known about plant lipocalins. We have previously reported the cloning of the first true plant lipocalins. Here we report the identification and characterization of plant lipocalins and lipocalin-like proteins using an integrated approach of data mining, expression studies, cellular localization, and phylogenetic analyses. Plant lipocalins can be classified into two groups, temperature-induced lipocalins (TILs) and chloroplastic lipocalins (CHLs). In addition, violaxanthin de-epoxidases (VDEs) and zeaxanthin epoxidases (ZEPs) can be classified as lipocalin-like proteins. CHLs, VDEs, and ZEPs possess transit peptides that target them to the chloroplast. On the other hand, TILs do not show any targeting peptide, but localization studies revealed that the proteins are found at the plasma membrane. Expression analyses by quantitative real-time PCR showed that expression of the wheat (Triticum aestivum) lipocalins and lipocalin-like proteins is associated with abiotic stress response and is correlated with the plant's capacity to develop freezing tolerance. In support of this correlation, data mining revealed that lipocalins are present in the desiccation-tolerant red algae Porphyra yezoensis and the cryotolerant marine yeast Debaryomyces hansenii, suggesting a possible association with stress-tolerant organisms. Considering the plant lipocalin properties, tissue specificity, response to temperature stress, and their association with chloroplasts and plasma membranes of green leaves, we hypothesize a protective function of the photosynthetic system against temperature stress. Phylogenetic analyses suggest that TIL lipocalin members in higher plants were probably inherited from a bacterial gene present in a primitive unicellular eukaryote. On the other hand, CHLs, VDEs, and ZEPs may have evolved from a cyanobacterial ancestral gene after the formation of the cyanobacterial endosymbiont from which the chloroplast originated.

TaVRT-2, a member of the StMADS-11 clade of flowering repressors, is regulated by vernalization and photoperiod in wheat.

The initiation of the reproductive phase in winter cereals is delayed during winter until favorable growth conditions resume in the spring. This delay is modulated by low temperature through the process of vernalization. The molecular and genetic bases of the interaction between environmental factors and the floral transition in these species are still unknown. However, the recent identification of the wheat (Triticum aestivum L.) TaVRT-1 gene provides an opportunity to decipher the molecular basis of the flowering-time regulation in cereals. Here, we describe the characterization of another gene, named TaVRT-2, possibly involved in the flowering pathway in wheat. Molecular and phylogenetic analyses indicate that the gene encodes a member of the MADS-box transcription factor family that belongs to a clade responsible for flowering repression in several species. Expression profiling of TaVRT-2 in near-isogenic lines and different genotypes with natural variation in their response to vernalization and photoperiod showed a strong relationship with floral transition. Its expression is up-regulated in the winter genotypes during the vegetative phase and in photoperiod-sensitive genotypes during short days, and is repressed by vernalization to a level that allows the transition to the reproductive phase. Protein-protein interaction studies revealed that TaVRT-2 interacts with proteins encoded by two important vernalization genes (TaVRT-1/VRN-1 and VRN-2) in wheat. These results support the hypothesis that TaVRT-2 is a putative repressor of the floral transition in wheat.

Molecular characterization and origin of novel bipartite cold-regulated ice recrystallization inhibition proteins from cereals.

To understand the molecular basis of freezing tolerance in plants, several low temperature-responsive genes have been identified from wheat. Among these are two genes named TaIRI-1 and TaIRI-2 (Triticum aestivum ice recrystallization inhibition) that are up-regulated during cold acclimation in freezing-tolerant species. Phytohormones involved in pathogen defense pathways (jasmonic acid and ethylene) induce the expression of one of the two genes. The encoded proteins are novel in that they have a bipartite structure that has never been reported for antifreeze proteins. Their N-terminal part shows similarity with the leucine-rich repeat-containing regions present in the receptor domain of receptor-like protein kinases, and their C-terminus is homologous to the ice-binding domain of some antifreeze proteins. The recombinant TaIRI-1 protein inhibits the growth of ice crystals, confirming its function as an ice recrystallization inhibition protein. The TaIRI genes were found only in the species belonging to the Pooideae subfamily of cereals. Comparative genomic analysis suggested that molecular evolutionary events took place in the genome of freezing-tolerant cereals to give rise to these genes with putative novel functions. These apparent adaptive DNA rearrangement events could be part of the molecular mechanisms that ensure the survival of hardy cereals in the harsh freezing environments.

Characterization of a calcium-soluble protein fraction from yellow mustard (Sinapis alba) seed meal with potential application as an additive to calcium-rich drinks.

A calcium-soluble protein isolate (CSPI) was prepared from the supernatant obtained after addition of 0.75 M calcium chloride to a pH 5.0 aqueous extract of yellow mustard (Sinapis alba) seed meal. Total amino acid analysis showed that the CSPI has significantly higher (p < 0.05) contents of glutamic acid + glutamine, cysteine, and proline when compared to the precipitated, calcium-insoluble proteins. Peptide mass fingerprinting of tryptic peptides of the major polypeptides by mass spectrometry indicated that the CSPI is composed mainly of cruciferin proteins with a contribution from napins (the major allergenic proteins of S. alba). The S. alba CSPI had significantly higher (p < 0.05) protein solubility and emulsion formation ability in the presence of 0.75 M calcium chloride when compared to similar isolates prepared from Brassica juncea (brown mustard) and soybean seed meals. We suggest that the S. alba CSPI could be used to prepare calcium-fortified high protein liquid products. However, the presence of allergenic proteins in this extract may limit its widespread food use.