Mario Houde

Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat

Expression of the acidic dehydrin gene wcor410 was found to be associated with the development of freezing tolerance in several Gramineae species. This gene is part of a family of three homologous members, wcor410, wcor410b, and wcor410c, that have been mapped to the long arms of the homologous group 6 chromosomes of hexaploid wheat. To gain insight into the function of this gene family, antibodies were raised against the WCOR410 protein and affinity purified to eliminate cross-reactivity with the WCS120 dehydrin-like protein of wheat. Protein gel blot analyses showed that the accumulation of WCOR410 proteins correlates well with the capacity of each cultivar to cold acclimate and develop freezing tolerance. Immunoelectron microscope analyses revealed that these proteins accumulate in the vicinity of the plasma membrane of cells in the sensitive vascular transition area where freeze-induced dehydration is likely to be more severe. Biochemical fractionation experiments indicated that WCOR410 is a peripheral protein and not an integral membrane protein. These results provide direct evidence that a subtype of the dehydrin family accumulates near the plasma membrane. The properties, abundance, and localization of these proteins suggest that they are involved in the cryoprotection of the plasma membrane against freezing or dehydration stress. We propose that WCOR410 plays a role in preventing the destabilization of the plasma membrane that occurs during dehydrative conditions.

The CBF gene family in hexaploid wheat and its relationship to the phylogenetic complexity of cereal CBFs.

Most temperate plants tolerate both chilling and freezing temperatures whereas many species from tropical regions suffer chilling injury when exposed to temperatures slightly above freezing. Cold acclimation induces the expression of cold-regulated genes needed to protect plants against freezing stress. This induction is mediated, in part, by the CBF transcription factor family. To understand the evolution and function of this family in cereals, we identified and characterized 15 different CBF genes from hexaploid wheat. Our analyses reveal that wheat species, T. aestivum and T. monococcum, may contain up to 25 different CBF genes, and that Poaceae CBFs can be classified into 10 groups that share a common phylogenetic origin and similar structural characteristics. Six of these groups (IIIc, IIId, IVa, IVb, IVc and IVd) are found only in the Pooideae suggesting they represent the CBF response machinery that evolved recently during colonization of temperate habitats. Expression studies reveal that five of the Pooideae-specific groups display higher constitutive and low temperature inducible expression in the winter cultivar, and a diurnal regulation pattern during growth at warm temperature. The higher constitutive and inducible expression within these CBF groups is an inherited trait that may play a predominant role in the superior low temperature tolerance capacity of winter cultivars and possibly be a basis of genetic variability in freezing tolerance within the Pooideae subfamily.

Differential expression of a gene encoding an acidic dehydrin in chilling sensitive and freezing tolerant gramineae species

We have characterized a new wheat cold‐regulated cDNA clone, Wcor410, that accumulates to equivalent levels in root, crown and leaf tissues during cold acclimation. The Wcor410 cDNA contains an ORF encoding a dehydrin‐like glutamate‐rich protein of 28 kDa with a pI of 5.1. However, the acidic nature, the absence of the glycine‐rich repeat and of the conserved N‐terminal region, DEYGNP, suggest that Wcor410 belongs to a different subgroup of the D11 protein family. Northern analysis showed that this gene is expressed only in freezing tolerant gramineae, whereas Southern analysis showed that the Wcor410 gene is present in all monocot species tested. The presence of freezing tolerance‐associated genes in sensitive species such as rice and corn is interesting. Characterization of the regulatory factors controlling these genes may help to establish an appropriate strategy to improve freezing tolerance.

A molecular marker to select for freezing tolerance in Gramineae

SummaryWe isolated, and expressed in Escherichia coli, a gene (Wcs120) that is strongly induced during cold acclimation of wheat. The gene product was purified and used to produce antibodies. Immunoblotting experiments with the anti-WCS120 antibody identified several cold-induced proteins named FTMs for Freezing Tolerance Markers since they are associated with the development of freezing tolerance. This protein family was found to be coordinately regulated specifically by low temperature, highly hydrophilic, stable to boiling, and to have a pI above 6.5. The accumulation kinetics during the acclimation period indicated a positive correlation with the capacity of each genotype to develop freezing tolerance. Accumulation of the proteins was higher in the freezing-tolerant genotype than in the less tolerant one. In addition, their accumulation was more pronounced in the crown and leaf tissues compared with roots, confirming a relationship to the capacity of the different tissues to develop freezing tolerance. Analysis of different species (eight monocots and four dicots) indicated that this protein family is specific for freezing-tolerant cereals. The antibody did not cross-react with any of the non-cereal species examined. The anti-FTMs antibody represents a potential tool for breeders to select for freezing tolerance traits in the Gramineae.

Structure and Functional Analysis of Wheat ICE (Inducer of CBF Expression) Genes

Two different inducers of CBF expression (ICE1-like genes), TaICE41 and TaICE87, were isolated from a cDNA library prepared from cold-treated wheat aerial tissues. TaICE41 encodes a protein of 381 aa with a predicted MW of 39.5 kDa while TaICE87 encodes a protein of 443 aa with a predicted MW of 46.5 kDa. TaICE41 and TaICE87 share 46% identity while they share 50 and 47% identity with Arabidopsis AtICE1 respectively. Expression analysis revealed that mRNA accumulation was not altered by cold treatment suggesting that both genes are expressed constitutively. Gel mobility shift analysis showed that TaICE41 and TaICE87 bind to different MYC elements in the wheat TaCBFIVd-B9 promoter. Transient expression assays in Nicotiana benthamiana, showed that both TaICE proteins can activate TaCBFIVd-B9 transcription. The different affinities of TaICE41 and TaICE87 for MYC variants suggest that ICE binding specificity may be involved in the differential expression of wheat CBF genes. Furthermore, analysis of MYC elements demonstrates that a specific variant is present in the wheat CBF group IV that is associated with freezing tolerance. Overexpression of either TaICE41 or TaICE87 genes in Arabidopsis enhanced freezing tolerance only upon cold acclimation suggesting that other factors induced by low temperature are required for their activity. The increased freezing tolerance in transgenic Arabidopsis is associated with a higher expression of the cold responsive activators AtCBF2, AtCBF3, and of several cold-regulated genes.

Isolation and characterization of wheat aluminum-regulated genes: possible involvement of aluminum as a pathogenesis response elicitor

Abstract. Using differential screening of a root tip cDNA library prepared from an Al-tolerant wheat cultivar (Triticum aestivum L. cv. Atlas-66) exposed to Al, we have isolated and characterized several wheat aluminum-regulated (War) cDNAs. Sequence comparison revealed that genes up-regulated by Al correspond to peroxidase (war4.2), cysteine proteinase (war5.2), phenylalanine-ammonia lyase (war7.2), and oxalate oxidase (war13.2). Two wheat cultivars that differ in their level of tolerance (cv. Atlas-66: tolerant, and cv. Fredrick: sensitive) were used to evaluate the relationship between the accumulation of War mRNAs and Al toxicity, as measured by root growth inhibition (RGI). The mRNA accumulation was modulated to similar levels in both cultivars compared at equivalent RGIs. This indicates that War mRNA accumulation is associated with the toxicity of Al rather than with the cultivars tolerance. It appears that most of the genes found to be up-regulated by Al share homologies with genes induced by pathogens. This suggests that Al may act as an elicitor of a pathogenesis-related transduction pathway. The potential functions of the up-regulated war genes in cell wall strengthening and Al trapping are discussed.

A leaf-specific gene stimulated by light during wheat acclimation to low temperature

We report here the identification and characterization of a new leaf-specific light-stimulated gene induced during cold acclimation of wheat. Sequence analysis revealed that the gene encodes a protein of 19 kDa with a pI of 8.8. This is a novel protein with a particular charge distribution. The C-terminal half has a high propensity to form an α-helix and contains all the acidic amino acids with a net negative charge of -7. On the other hand, the N-terminal half is rich in proline, lysine and arginine with a net positive charge of +10. These properties are commonly found in several transcription factors. The protein is also rich in alanine (21%), is hydrophilic but not boiling soluble in contrast to other alanine-rich proteins. During low temperature exposure, the corresponding mRNA accumulates rapidly in the leaf and remains at a constant level in two tolerant cultivars used. However, is a less tolerant cultivar, the mRNA level declines despite maintaining the plants at 4°C. Southern blot analysis indicates that the differential expression in the less tolerant genotype is not due to a different genomic organization or gene copy number. The mRNA was specifically localized in leaf tissues and increased several-fold during the greening at 4°C. Furthermore, this gene is not induced in callus cultures acclimated in the absence or presence of light. This suggests that the full expression of this gene is dependent on organized leaf tissue. The expression of this gene was not affected by ABA, drought, heat shock, salinity, wounding or anaerobiosis, demonstrating that it is specifically induced by low temperature. The Wcs19 mRNA is preferentially expressed in tolerant Gramineae species.

Identification of genes and pathways associated with aluminum stress and tolerance using transcriptome profiling of wheat near-isogenic lines

BackgroundAluminum is considered the most limiting factor for plant productivity in acidic soils, which cover large areas of the worlds potential arable lands. The inhibition of root growth is recognized as the primary effect of Al toxicity. To identify genes associated with Al stress and tolerance, transcriptome analyses of four different wheat lines (2 Al-tolerant and 2 Al sensitive) that differ in their response to Al were performed.ResultsMicroarray expression profiling revealed that 83 candidate genes are associated with Al stress and 25 are associated with tolerance. The stress-associated genes include important enzymes such as pyruvate dehydrogenase, alternative oxidase, and galactonolactone oxidase, ABC transporter and ascorbate oxido-reducatase. The Al tolerance-associated genes include the ALMT-1 malate transporter, glutathione S-transferase, germin/oxalate oxidase, fructose 1,6-bisphosphatase, cysteine-rich proteins, cytochrome P450 monooxygenase, cellulose synthase, zinc finger transcription factor, disease resistance response protein and F-box containing domain protein.ConclusionIn this survey, we identified stress- and tolerance-associated genes that may be involved in the detoxification of Al and reactive oxygen species. Alternative pathways could help maintain the supply of important metabolites (H2O2, ascorbate, NADH, and phosphate) needed for Al tolerance and root growth. The Al tolerance-associated genes may be key factors that regulate these pathways.

The plant Apolipoprotein D ortholog protects Arabidopsis against oxidative stress

BackgroundLipocalins 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.ResultsOur 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.ConclusionThis 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.

Caco-2 Cell Line Used as an In Vitro Model to Study Cadmium Accumulation in Intestinal Epithelial Cells

Abstract.109Cd uptake was studied using the highly differentiated TC7 clone of Caco-2 cells as a model of human enterocyte function. Intracellular accumulation of 0.3 μm109Cd involved a rapid and a slow uptake phase, which resulted in complete equilibration (t½= 17.3 ± 1.3 min) with an apparent in-to-out distribution ratio (αe) of 11.6 ± 0.8. The amplitude of the rapid phase (U0) and the rate of the slow phase (V) were similarly reduced in the less differentiated PF11 clone, but comparable αe values were observed at equilibrium. In both clones, the t½ and αe values increased and decreased, respectively, upon addition of unlabeled Cd to the uptake media. In TC7 cells, 109Cd uptake at 1 min (U1) was unaffected by Ca concentrations four order of magnitude in excess, but both U0 and V demonstrated similar sensitivities to unlabeled Cd, Zn and sulfhydryl-reactive agents. Only U0 disappeared when EDTA was present in the wash solutions. U1 showed saturation kinetics and the data were found compatible with a model assuming rapid initial Cd binding and transport through a unique transport protein (Km= 3.8 ± 0.7 μm). Cd efflux kinetics demonstrated partial reversibility in EDTA-containing solutions, suggesting that the taken up Cd might be both tightly and loosely bound to intracellular binding sites. However, the displacement of 109Cd measured at 65 min failed to reveal this heterogeneity: the data were found compatible with a model equation assuming the presence of one class of high-capacity high-affinity binding sites. We conclude that a slow-transport fast-intracellular binding mechanism of Cd uptake best accounts for these results and that Cd transport most likely involves a carrier-type of protein unrelated to Ca absorption.