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.

Wheat CBF gene family: identification of polymorphisms in the CBF coding sequence.

Expression of cold-regulated genes needed for protection against freezing stress is mediated, in part, by the CBF transcription factor family. Previous studies with temperate cereals suggested that the CBF gene family in wheat was large, and that CBF genes were at the base of an important low temperature tolerance trait. Therefore, the goal of our study was to identify the CBF repertoire in the freezing-tolerant hexaploid wheat cultivar Norstar, and then to examine if the coding region of CBF genes in two spring cultivars contain polymorphisms that could affect the protein sequence and structure. Our analyses reveal that hexaploid wheat contains a complex CBF family consisting of at least 65 CBF genes of which 60 are known to be expressed in the cultivar Norstar. They represent 27 paralogous genes with 1-3 homeologous copies for the A, B, and D genomes. The cultivar Norstar contains two pseudogenes and at least 24 additional proteins having sequences and (or) structures that deviate from the consensus in the conserved AP2 DNA-binding and (or) C-terminal activation-domains. This suggests that in cultivars such as Norstar, low temperature tolerance may be increased through breeding of additional optimal alleles. The examination of the CBF repertoire present in the two spring cultivars, Chinese Spring and Manitou, reveals that they have additional polymorphisms affecting conserved positions in these domains. Understanding the effects of these polymorphisms will provide additional information for the selection of optimum CBF alleles in Triticeae breeding programs.

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.

Regulatory gene candidates and gene expression analysis of cold acclimation in winter and spring wheat.

Freezing tolerance in plants develops through acclimation to cold by growth at low, above-freezing temperatures. Wheat is one of the most freezing-tolerant plants among major crop species and the wide range of freezing tolerance among wheat cultivars makes it an excellent model for investigation of the genetic basis of cold tolerance. Large numbers of genes are known to have altered levels of expression during the period of cold acclimation and there is keen interest in deciphering the signaling and regulatory pathways that control the changes in gene expression associated with acquired freezing tolerance. A 5740 feature cDNA amplicon microarray that was enriched for signal transduction and regulatory genes was constructed to compare changes in gene expression in a highly cold-tolerant winter wheat cultivar CDC Clair and a less tolerant spring cultivar, Quantum. Changes in gene expression over a time course of 14 days detected over 450 genes that were regulated by cold treatment and were differentially regulated between spring and winter cultivars, of these 130 are signaling or regulatory gene candidates, including: transcription factors, protein kinases, ubiquitin ligases and GTP, RNA and calcium binding proteins. Dynamic changes in transcript levels were seen at all periods of cold acclimation in both cultivars. There was an initial burst of gene activity detectable during the first day of CA, during which 90% of all genes with increases in transcript levels became clearly detectable and early expression differential between the two cultivars became more disparate with each successive period of cold acclimation.

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.

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.

Transcriptome comparison of winter and spring wheat responding to low temperature.

Freezing tolerance in plants is a complex trait that occurs in many plant species during growth at low, nonfreezing temperatures, a process known as cold acclimation. This process is regulated by a multigenic system expressing broad variation in the degree of freezing tolerance among wheat cultivars. Microarray analysis is a powerful and rapid approach to gene discovery. In species such as wheat, for which large scale mutant screening and transgenic studies are not currently practical, genotype comparison by this methodology represents an essential approach to identifying key genes in the acquisition of freezing tolerance. A microarray was constructed with PCR amplified cDNA inserts from 1184 wheat expressed sequence tags (ESTs) that represent 947 genes. Gene expression during cold acclimation was compared in 2 cultivars with marked differences in freezing tolerance. Transcript levels of more than 300 genes were altered by cold. Among these, 65 genes were regulated differently between the 2 cultivars for at least 1 time point. These include genes that encode potential regulatory proteins and proteins that act in plant metabolism, including protein kinases, putative transcription factors, Ca2+ binding proteins, a Golgi localized protein, an inorganic pyrophosphatase, a cell wall associated hydrolase, and proteins involved in photosynthesis.

TaVRT-1, a putative transcription factor associated with vegetative to reproductive transition in cereals.

The molecular genetics of vernalization, defined as the promotion of flowering by cold treatment, is still poorly understood in cereals. To better understand this mechanism, we cloned and characterized a gene that we named TaVRT-1 (wheat [Triticum aestivum] vegetative to reproductive transition-1). Molecular and sequence analyses indicated that this gene encodes a protein homologous to the MADS-box family of transcription factors that comprises certain flowering control proteins in Arabidopsis. Mapping studies have localized this gene to the Vrn-1 regions on the long arms of homeologous group 5 chromosomes, regions that are associated with vernalization and freezing tolerance (FT) in wheat. The level of expression of TaVRT-1 is positively associated with the vernalization response and transition from vegetative to reproductive phase and is negatively associated with the accumulation of COR genes and degree of FT. Comparisons among different wheat genotypes, near-isogenic lines, and cereal species, which differ in their vernalization response and FT, indicated that the gene is inducible only in those species that require vernalization, whereas it is constitutively expressed in spring habit genotypes. In addition, experiments using both the photoperiod-sensitive barley (Hordeum vulgare cv Dicktoo) and short or long day de-acclimated wheat revealed that the expression of TaVRT-1 is also regulated by photoperiod. These expression studies indicate that photoperiod and vernalization may regulate this gene through separate pathways. We suggest that TaVRT-1 is a key developmental gene in the regulatory pathway that controls the transition from the vegetative to reproductive phase in cereals.

Expression profiling and bioinformatic analyses of a novel stress-regulated multispanning transmembrane protein family from cereals and Arabidopsis.

Cold acclimation is a multigenic trait that allows hardy plants to develop efficient tolerance mechanisms needed for winter survival. To determine the genetic nature of these mechanisms, several cold-responsive genes of unknown function were identified from cold-acclimated wheat (Triticum aestivum). To identify the putative functions and structural features of these new genes, integrated genomic approaches of data mining, expression profiling, and bioinformatic predictions were used. The analyses revealed that one of these genes is a member of a small family that encodes two distinct groups of multispanning transmembrane proteins. The cold-regulated (COR)413-plasma membrane and COR413-thylakoid membrane groups are potentially targeted to the plasma membrane and thylakoid membrane, respectively. Further sequence analysis of the two groups from different plant species revealed the presence of a highly conserved phosphorylation site and a glycosylphosphatidylinositol-anchoring site at the C-terminal end. No homologous sequences were found in other organisms suggesting that this family is specific to the plant kingdom. Intraspecies and interspecies comparative gene expression profiling shows that the expression of this gene family is correlated with the development of freezing tolerance in cereals and Arabidopsis. In addition, several members of the family are regulated by water stress, light, and abscisic acid. Structure predictions and comparative genome analyses allow us to propose that the cor413 genes encode putative G-protein-coupled receptors.

Cold-regulated cereal chloroplast late embryogenesis abundant-like proteins. Molecular characterization and functional analyses.

Cold acclimation and freezing tolerance are the result of complex interaction between low temperature, light, and photosystem II (PSII) excitation pressure. Previous results have shown that expression of the Wcs19 gene is correlated with PSII excitation pressure measured in vivo as the relative reduction state of PSII. Using cDNA library screening and data mining, we have identified three different groups of proteins, late embryogenesis abundant (LEA) 3-L1, LEA3-L2, and LEA3-L3, sharing identities with WCS19. These groups represent a new class of proteins in cereals related to group 3 LEA proteins. They share important characteristics such as a sorting signal that is predicted to target them to either the chloroplast or mitochondria and a C-terminal sequence that may be involved in oligomerization. The results of subcellular fractionation, immunolocalization by electron microscopy and the analyses of target sequences within the Wcs19 gene are consistent with the localization of WCS19 within the chloroplast stroma of wheat (Triticum aestivum) and rye (Secale cereale). Western analysis showed that the accumulation of chloroplastic LEA3-L2 proteins is correlated with the capacity of different wheat and rye cultivars to develop freezing tolerance. Arabidopsis was transformed with the Wcs19 gene and the transgenic plants showed a significant increase in their freezing tolerance. This increase was only evident in cold-acclimated plants. The putative function of this protein in the enhancement of freezing tolerance is discussed.

Molecular and biochemical characterization of a cold-regulated phosphoethanolamine N-methyltransferase from wheat.

A cDNA that encodes a methyltransferase (MT) was cloned from a cold-acclimated wheat (Triticum aestivum) cDNA library. Molecular analysis indicated that the enzyme WPEAMT (wheat phosphoethanolamine [P-EA] MT) is a bipartite protein with two separate sets of S-adenosyl-L-Met-binding domains, one close to the N-terminal end and the second close to the C-terminal end. The recombinant protein was found to catalyze the three sequential methylations of P-EA to form phosphocholine, a key precursor for the synthesis of phosphatidylcholine and glycine betaine in plants. Deletion and mutation analyses of the two S-adenosyl-L-Met-binding domains indicated that the N-terminal domain could perform the three N-methylation steps transforming P-EA to phosphocholine. This is in contrast to the MT from spinach (Spinacia oleracea), suggesting a different functional evolution for the monocot enzyme. The truncated C-terminal and the N-terminal mutated enzyme were only able to methylate phosphomonomethylethanolamine and phosphodimethylethanolamine, but not P-EA. This may suggest that the C-terminal part is involved in regulating the rate and the equilibrium of the three methylation steps. Northern and western analyses demonstrated that both Wpeamt transcript and the corresponding protein are up-regulated during cold acclimation. This accumulation was associated with an increase in enzyme activity, suggesting that the higher activity is due to de novo protein synthesis. The role of this enzyme during cold acclimation and the development of freezing tolerance are discussed.