Duplicated antagonistic EPF peptides optimize grass stomatal initiation.

Peptide signaling has emerged as a key component of plant growth and development, including stomatal patterning, which is crucial for plant productivity and survival. Although exciting progress has been made in understanding EPIDERMAL PATTERNING FACTOR (EPF) signaling in Arabidopsis, the mechanisms by which EPF peptides control different stomatal patterns and morphologies in grasses are poorly understood. Here, by examining expression patterns, overexpression transgenics and cross-species complementation, the antagonistic stomatal ligands orthologous to Arabidopsis AtEPF2 and AtSTOMAGEN/AtEPFL9 peptides were identified in Triticum aestivum (wheat) and the grass model organism Brachypodium distachyon. Application of bioactive BdEPF2 peptides inhibited stomatal initiation, but not the progression or differentiation of stomatal precursors in Brachypodium. Additionally, the inhibitory roles of these EPF peptides during grass stomatal development were suppressed by the contrasting positive action of the BdSTOMAGEN peptide in a dose-dependent manner. These results not only demonstrate how conserved EPF peptides that control different stomatal patterns exist in nature, but also suggest new strategies to improve crop yield through the use of plant-derived antagonistic peptides that optimize stomatal density on the plant epidermis.

Characterization and expression of the Pirin gene family in Triticum aestivum.

Pirins are nuclear bicupin proteins, encoded by genes that are one of several gene families that comprise the cupin superfamily in plants. Pirin genes have been implicated in stress response pathways studied in Arabidopsis and At-Pirin1 has been shown to interact with the heterotrimeric G-protein alpha subunit (GPA1). The aim of this study was to identify the members of the Pirin gene family in Triticum aestivum, to correct their annotations in the whole genome, and gain an insight into their tissue-specific expression as well as their response to abiotic and biotic stresses. The Pirin gene family in T. aestivum is comprised of 18 genes that represent six paralogous gene copies, each having an A, B, and D homeolog. Expression analysis of the Pirin genes in T. aestivum Illumina RNA-seq libraries, which included sampling from differing tissue types as well as abiotic and biotic stresses, indicates that the members of the Pirin gene family have specialized expression and play a role in stress responses. Pirin gene families are also identified in other monocots including Aegilops tauschii, Hordeum vulgare, Brachypodium distachyon, Oryza sativa, Zea mays, Sorghum bicolor, and the dicot Arabidopsis thaliana.

The stress induced caleosin, RD20/CLO3, acts as a negative regulator of GPA1 in Arabidopsis.

KEY MESSAGE: A stress induced calcium-binding protein, RD20/CLO3 interacts with the alpha subunit of the heterotrimeric G-protein complex in Arabidopsis and affects etiolation and leaf morphology. Heterotrimeric G proteins and calcium signaling have both been shown to play a role in the response to environmental abiotic stress in plants; however, the interaction between calcium-binding proteins and G-protein signaling molecules remains elusive. We investigated the interaction between the alpha subunit of the heterotrimeric G-protein complex, GPA1, of Arabidopsis thaliana with the calcium-binding protein, the caleosin RD20/CLO3, a gene strongly induced by drought, salt and abscisic acid. The proteins were found to interact in vivo by bimolecular fluorescent complementation (BiFC); the interaction was localized to the endoplasmic reticulum and to oil bodies within the cell. The constitutively GTP-bound GPA1 (GPA1QL) also interacts with RD20/CLO3 as well as its EF-hand mutant variations and these interactions are localized to the plasma membrane. The N-terminal portion of RD20/CLO3 was found to be responsible for the interaction with GPA1 and GPA1QL using both BiFC and yeast two-hybrid assays. RD20/CLO3 contains a single calcium-binding EF-hand in the N-terminal portion of the protein; disruption of the calcium-binding capacity of the protein obliterates interaction with GPA1 in in vivo assays and decreases the interaction between the caleosin and the constitutively active GPA1QL. Analysis of rd20/clo3 mutants shows that RD20/CLO3 plays a key role in the signaling pathway controlling hypocotyl length in dark grown seedlings and in leaf morphology. Our findings indicate a novel role for RD20/CLO3 as a negative regulator of GPA1.

Structural variation and rates of genome evolution in the grass family seen through comparison of sequences of genomes greatly differing in size.

Homology was searched with genes annotated in the Aegilops tauschii pseudomolecules against genes annotated in the pseudomolecules of tetraploid wild emmer wheat, Brachypodium distachyon, sorghum and rice. Similar searches were performed with genes annotated in the rice pseudomolecules. Matrices of collinear genes and rearrangements in their order were constructed. Optical BioNano genome maps were constructed and used to validate rearrangements unique to the wild emmer and Ae. tauschii genomes. Most common rearrangements were short paracentric inversions and short intrachromosomal translocations. Intrachromosomal translocations outnumbered segmental intrachromosomal duplications. The densities of paracentric inversion lengths were approximated by exponential distributions in all six genomes. Densities of collinear genes along the Ae. tauschii chromosomes were highly correlated with meiotic recombination rates but those of rearrangements were not, suggesting different causes of the erosion of gene collinearity and evolution of major chromosome rearrangements. Frequent rearrangements sharing breakpoints suggested that chromosomes have been rearranged recurrently at some sites. The distal 4 Mb of the short arms of rice chromosomes Os11 and Os12 and corresponding regions in the sorghum, B. distachyon and Triticeae genomes contain clusters of interstitial translocations including from 1 to 7 collinear genes. The rates of acquisition of major rearrangements were greater in the large wild emmer wheat and Ae. tauschii genomes than in the lineage preceding their divergence or in the B. distachyon, rice and sorghum lineages. It is suggested that synergy between large quantities of dynamic transposable elements and annual growth habit have been the primary causes of the fast evolution of the Triticeae genomes.

Characterization of the Esi3/RCI2/PMP3 gene family in the Triticeae.

BACKGROUND: Members of the Early Salt Induced 3 (Esi3/RCI2/PMP3) gene family in plants have been shown to be induced in response to both biotic and abiotic stresses and to enhance stress tolerance in both transgenic plants and Saccharomyces cerevisiae. Esi3 was first identified as a salt stress induced gene in the salt tolerant wild wheat grass, Lophopyrum elongatum, and subsequently homologous genes in many other species were found to be members of the gene family. These include Arabidopsis thaliana and Oryza sativa where they are referred to as Rare Cold Inducible 2 (RCI2), and Zea mays where they are referred to as Plasma Membrane Protein 3 (PMP3). This study characterizes the Esi3 family members in Triticum aestivum and explores the tissue specific expression patterns of the gene family members as well as their response to a variety of environmental stresses. RESULTS: The Esi3 gene family was found to have a total of 29 family members comprised of ten paralogous groups in the hexaploid T. aestivum. Each paralogous group contains three homeologous copies, one in each of the A, B and D genomes with the exception of Esi3-2 which is missing the B copy. The genes of the Esi3 gene family were also identified in four other monocot species, Aegilops tauschii, Hordeum vulgare, Secale cereale and Sorghum bicolor, and were confirmed or corrected for Brachypodium distachyon, Oryza sativa and Zea mays, as well as the dicot Arabidopsis thaliana. Gene expression of the Esi3s was analyzed using tissue-specific, abiotic and biotic stress RNA-Seq 454 sequence libraries and Affymetrix microarray data for T. aestivum. CONCLUSIONS: Members of nearly all paralogous groups of the Esi3 genes in T. aestivum have altered gene expression in response to abiotic or biotic stress conditions. In addition, there are modest differences in gene expression among homeologous members of the gene family. This suggests that the Esi3 gene family plays an important role in the plants response to the stresses presented in this study. The Esi3-9 in T. aestivum has a unique N terminal extension placing it into Group III, a new group for the Esi3/RCI2/PMP3 gene family.

Identification and characterization of rye genes not expressed in allohexaploid triticale.

BACKGROUND: One of the most important evolutionary processes in plants is polyploidization. The combination of two or more genomes in one organism often initially leads to changes in gene expression and extensive genomic reorganization, compared to the parental species. Hexaploid triticale (x Triticosecale) is a synthetic hybrid crop species generated by crosses between T. turgidum and Secale cereale. Because triticale is a recent synthetic polyploid it is an important model for studying genome evolution following polyploidization. Molecular studies have demonstrated that genomic sequence changes, consisting of sequence elimination or loss of expression of genes from the rye genome, are common in triticale. High-throughput DNA sequencing allows a large number of genes to be surveyed, and transcripts from the different homeologous copies of the genes that have high sequence similarity can be better distinguished than hybridization methods previously employed. RESULTS: The expression levels of 23,503 rye cDNA reference contigs were analyzed in 454-cDNA libraries obtained from anther, root and stem from both triticale and rye, as well as in five 454-cDNA data sets created from triticale seedling shoot, ovary, stigma, pollen and seed tissues to identify the classes of rye genes silenced or absent in the recent synthetic hexaploid triticale. Comparisons between diploid rye and hexaploid triticale detected 112 rye cDNA contigs (~0.5%) that were totally undetected by expression analysis in all triticale tissues, although their expression was relatively high in rye tissues. Non-expressed rye genes were found to be strikingly less similar to their closest BLASTN matches in the wheat genome or in the other Triticum genomes than a test set of 200 random rye genes. Genes that were not detected in the RNA-seq data were further characterized by testing for their presence in the triticale genome by PCR using genomic DNA as a template. CONCLUSION: Genes with low similarity between rye sequences and their closest matches in the Triticum genome have a higher probability to be repressed or absent in the allopolyploid genome.

Characterization of the caleosin gene family in the Triticeae.

BACKGROUND: The caleosin genes encode proteins with a single conserved EF hand calcium-binding domain and comprise small gene families found in a wide range of plant species. Some members of the gene family have been shown to be upregulated by environmental stresses including low water availability and high salinity. Caleosin 3 from wheat has been shown to interact with the α-subunit of the heterotrimeric G proteins, and to act as a GTPase activating protein (GAP). This study characterizes the size and diversity of the gene family in wheat and related species and characterizes the differential tissue-specific expression of members of the gene family. RESULTS: A total of 34 gene family members that belong to eleven paralogous groups of caleosins were identified in the hexaploid bread wheat, T. aestivum. Each group was represented by three homeologous copies of the gene located on corresponding homeologous chromosomes, except the caleosin 10, which has four gene copies. Ten gene family members were identified in diploid barley, Hordeum vulgare, and in rye, Secale cereale, seven in Brachypodium distachyon, and six in rice, Oryza sativa. The analysis of gene expression was assayed in triticale and rye by RNA-Seq analysis of 454 sequence sets and members of the gene family were found to have diverse patterns of gene expression in the different tissues that were sampled in rye and in triticale, the hybrid hexaploid species derived from wheat and rye. Expression of the gene family in wheat and barley was also previously determined by microarray analysis, and changes in expression during development and in response to environmental stresses are presented. CONCLUSIONS: The caleosin gene family had a greater degree of expansion in the Triticeae than in the other monocot species, Brachypodium and rice. The prior implication of one member of the gene family in the stress response and heterotrimeric G protein signaling, points to the potential importance of the caleosin gene family. The complexity of the family and differential expression in various tissues and under conditions of abiotic stress suggests the possibility that caleosin family members may play diverse roles in signaling and development that warrants further investigation.

Gene expression analysis in the roots of salt-stressed wheat and the cytogenetic derivatives of wheat combined with the salt-tolerant wheatgrass, Lophopyrum elongatum.

KEY MESSAGE: Using microarray analysis, we identified regulatory and signaling-related genes with differential expression in three genotypes with varying degrees of salt tolerance, Triticum aestivum , the amphiploid, and the wheat substitution line DS3E(3A). Lophopyrum elongatum is among one of the most salt-tolerant members of the Triticeae; important genetic stocks developed from crosses between wheat and L. elongatum provide a unique opportunity to compare gene expression in response to salt stress between these highly related species. The octaploid amphiploid contains the entire genome of T. aestivum and L. elongatum, and the disomic substitution line DS3E(3A) has chromosome 3A of wheat replaced by chromosome 3E of L. elongatum. In this study, microarray analysis was used to characterize gene expression profiles in the roots of three genotypes, Triticum aestivum, the octaploid amphiploid, and the wheat DS3E(3A) substitution line, in response to salt stress. We first examined changes in gene expression in wheat over a time course of 3 days of salt stress, and then compared changes in gene expression in wheat, the T. aestivum × L. elongatum amphiploid, and in the DS3E(3A) substitution line after 3 days of salt stress. In the time course experiment, 237 genes had 1.5 fold or greater change at least one out of three time points assayed in the experiment. The comparison between the three genotypes revealed 304 genes with significant differences in changes of expression between the genotypes. Forty-two of these genes had at least a twofold change in expression in response to salt treatment; 18 of these genes have signaling or regulatory function. Genes with significant differences in induction or repression between genotypes included transcription factors, protein kinases, ubiquitin ligases and genes related to phospholipid signaling.

The caleosin RD20/CLO3 regulates lateral root development in response to abscisic acid and regulates flowering time in conjunction with the caleosin CLO7.

The caleosins are encoded by multi-gene families in Arabidopsis thaliana and other plant species. This work investigates the role of two family members, RD20/CLO3 and CLO7, in flowering transition and in root development in response to ABA treatment. Gene expression of the caleosin RD20/CLO3 is induced by ABA in the root tissues and RD20/CLO3 has a negative affect on the total number of lateral roots as well as the length of the lateral roots in response to ABA treatment. The rd20/clo3 mutant has more and longer lateral roots in response to ABA treatment compared to the wild-type, showing that RD20/CLO3 plays a role in the ABA signaling pathway affecting this trait. In contrast, the caleosin CLO7 is not expressed in the roots and does not affect root architecture in response to ABA treatment. The disruption of both RD20/CLO3 and CLO7 together causes a dramatic early-flowering phenotype under long-day conditions, whereas single mutations in these genes do not affect flowering time under these conditions. Both yeast two-hybrid and bimolecular fluorescence complementation showed that both RD20/CLO3 and CLO7 interact with each other and can form homodimers and heterodimers. Taken together, these findings suggest that members of the caleosin gene family play both different and redundant roles in plant development.

Characterization of the heterotrimeric G protein gene families in Triticum aestivum and related species.

This study characterizes the heterotrimeric G protein gene families in Triticum aestivum, their tissue-specific expression patterns during development and in response to biotic and abiotic stress conditions. There are three Gα genes, three Gß and 12 Gγ genes, totaling 18 genes encoding heterotrimeric G proteins in the hexaploid wheat genome. Each haploid genome of the hexaploid T. aestivum has a single gene encoding the α subunit of the heterotrimeric G protein complex, GA1, a single Gß and four Gγ genes. Each gene has three homeologous copies in the A, B and D genomes. The physical interaction between the Gß (Gpb) and two Gγ subunits, Gpg1 and Gpg2, was shown through bimolecular fluorescence complementation (BiFC). The gene expression in response to biotic and abiotic stresses showed both up-regulation and down-regulation of members of the gene families. Gγ2-B and Gγ2-D are significantly upregulated during heat stress, GA1-D is upregulated by cold stress and Gγ1-A and Gγ1-D were upregulated by Fusarium graminearum inoculation in a F. graminearum resistant cultivar. This suggests that these members may play roles in biotic and abiotic signaling pathways and the roles of these genes within these pathways need further investigation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03156-9.

The caleosin CLO7 and its role in the heterotrimeric G-protein signalling network.

The investigation of the caleosin CLO7 in relation to heterotrimeric G-protein signalling in Arabidopsis showed that the gene plays a role in seed germination and embryo viability. The caleosin CLO7 belongs to a multi-gene family of calcium-binding proteins which are characterized by single EF-hand motifs. Other members of the caleosin gene family have been shown to affect transpiration and seed germination as well as play a role in both abiotic and biotic stress responses. The proteins are associated with lipid droplets/oil bodies and some members of the gene family have been shown to have peroxygenase activity. Members of the gene family have also been shown to interact with the α subunit of the heterotrimeric G protein complex. In this study, we further expand on the diversity of physiological responses in which members of this gene family play regulatory roles. Utilizing BiFC and Y2H protein-protein interaction assays, CLO7 is identified as an interactor of the heterotrimeric G protein α subunit, GPA1. The full-length CLO7 is shown to interact with both the wild-type GPA1 and its constitutively active form, GPA1QL, at the plasma membrane. Point mutations to critical amino acids for calcium binding in the EF-hand of CLO7 indicate that the interaction with GPA1 is calcium-dependent and that the interaction with GPA1QL is enhanced by calcium. Protein-protein interaction assays also show that CLO7 interacts with Pirin1, a member of the cupin gene superfamily and a known downstream effector of GPA1, and this interaction is calcium-dependent. The N-terminal portion of CLO7 is responsible for these interactions. GFP-tagged CLO7 protein localizes to the endoplasmic reticulum (ER) and to lipid bodies. Characterization of the clo7 mutant line has shown that CLO7 is implicated in the abscisic acid (ABA) and mannitol-mediated inhibition of seed germination, with the clo7 mutant displaying higher germination rates in response to osmotic stress and ABA hormone treatment. These results provide insight into the role of CLO7 in seed germination in response to abiotic stress as well as its interaction with GPA1 and Pirin1. CLO7 also plays a role in embryo viability with the clo7gpa1 double mutant displaying embryo lethality, and therefore the double mutant cannot be recovered.

Heterotrimeric Gα subunit from wheat (Triticum aestivum), GA3, interacts with the calcium-binding protein, Clo3, and the phosphoinositide-specific phospholipase C, PI-PLC1.

The canonical Gα subunit of the heterotrimeric G protein complex from wheat (Triticum aestivum), GA3, and the calcium-binding protein, Clo3, were revealed to interact both in vivo and in vitro and Clo3 was shown to enhance the GTPase activity of GA3. Clo3 is a member of the caleosin gene family in wheat with a single EF-hand domain and is induced during cold acclimation. Bimolecular Fluorescent Complementation (BiFC) was used to localize the interaction between Clo3 and GA3 to the plasma membrane (PM). Even though heterotrimeric G-protein signaling and Ca²âº signaling have both been shown to play a role in the response to environmental stresses in plants, little is known about the interaction between calcium-binding proteins and Gα. The GAP activity of Clo3 towards GA3 suggests it may play a role in the inactivation of GA3 as part of the stress response in plants. GA3 was also shown to interact with the phosphoinositide-specific phospholipase C, PI-PLC1, not only in the PM but also in the endoplasmic reticulum (ER). Surprisingly, Clo3 was also shown to interact with PI-PLC1 in the PM and ER. In vitro analysis of the protein-protein interaction showed that the interaction of Clo3 with GA3 and PI-PLC1 is enhanced by high Ca²âº levels. Three-way affinity characterizations with GA3, Clo3 and PI-PLC1 showed the interaction with Clo3 to be competitive, which suggests that Clo3 may play a role in the Ca²âº-triggered feedback regulation of both GA3 and PI-PLC1. This hypothesis was further supported by the demonstration that Clo3 has GAP activity with GA3.

Aegilops tauschii genome assembly Aet v5.0 features greater sequence contiguity and improved annotation.

Aegilops tauschii is the donor of the D subgenome of hexaploid wheat and an important genetic resource. The reference-quality genome sequence Aet v4.0 for Ae. tauschii acc. AL8/78 was therefore an important milestone for wheat biology and breeding. Further advances in sequencing acc. AL8/78 and release of the Aet v5.0 sequence assembly are reported here. Two new optical maps were constructed and used in the revision of pseudomolecules. Gaps were closed with Pacific Biosciences long-read contigs, decreasing the gap number by 38,899. Transposable elements and protein-coding genes were reannotated. The number of annotated high-confidence genes was reduced from 39,635 in Aet v4.0 to 32,885 in Aet v5.0. A total of 2245 biologically important genes, including those affecting plant phenology, grain quality, and tolerance of abiotic stresses in wheat, was manually annotated and disease-resistance genes were annotated by a dedicated pipeline. Disease-resistance genes encoding nucleotide-binding site domains, receptor-like protein kinases, and receptor-like proteins were preferentially located in distal chromosome regions, whereas those encoding transmembrane coiled-coil proteins were dispersed more evenly along the chromosomes. Discovery, annotation, and expression analyses of microRNA (miRNA) precursors, mature miRNAs, and phasiRNAs are reported, including miRNA target genes. Other small RNAs, such as hc-siRNAs and tRFs, were characterized. These advances enhance the utility of the Ae. tauschii genome sequence for wheat genetics, biotechnology, and breeding.

Aegilops tauschii Genome Sequence: A Framework for Meta-analysis of Wheat QTLs.

Numerous quantitative trait loci (QTL) have been mapped in tetraploid and hexaploid wheat and wheat relatives, mostly with simple sequence repeat (SSR) or single nucleotide polymorphism (SNP) markers. To conduct meta-analysis of QTL requires projecting them onto a common genomic framework, either a consensus genetic map or genomic sequence. The latter strategy is pursued here. Of 774 QTL mapped in wheat and wheat relatives found in the literature, 585 (75.6%) were successfully projected onto the Aegilops tauschii pseudomolecules. QTL mapped with SNP markers were more successfully projected (92.2%) than those mapped with SSR markers (66.2%). The QTL were not distributed homogeneously along chromosome arms. Their frequencies increased in the proximal-to-distal direction but declined in the most distal regions and were weakly correlated with recombination rates along the chromosome arms. Databases for projected SSR markers and QTL were constructed and incorporated into the Ae. tauschii JBrowse. To facilitate meta-QTL analysis, eight clusters of QTL were used to estimate standard deviations ([Formula: see text]) of independently mapped QTL projected onto the Ae. tauschii genome sequence. The standard deviations [Formula: see text] were modeled as an exponential decay function of recombination rates along the Ae. tauschii chromosomes. We implemented four hypothesis tests for determining the membership of query QTL. The hypothesis tests and estimation procedure for [Formula: see text] were implemented in a web portal for meta-analysis of projected QTL. Twenty-one QTL for Fusarium head blight resistance mapped on wheat chromosomes 3A, 3B, and 3D were analyzed to illustrate the use of the portal for meta-QTL analyses.

Genetic combining ability of coriander genotypes for agronomic and phytochemical traits in response to contrasting irrigation regimes.

Knowledge of genetic combining ability and gene action would help breeders to choose suitable parents and devise an appropriate breeding strategy for coriander. In the present study, six diverse genotypes of coriander, their 15 F1s and 15 F2s were evaluated through randomized complete block design with three replications to study genetic combining ability for agronomic and phytochemical traits in coriander. Plants were subjected to well-watered (WW), mild water-deficit stress (MWDS) and severe water-deficit stress (SWDS) irrigation regimes. The results indicate that water-deficit stress decreased all of the measured traits in both the F1 and F2 generations. General combining ability and specific combining ability effects were highly significant for all of the traits in both the F1 and F2 generations. Additive gene action was predominant for phonology and fruit yield component traits in all irrigation regimes in both the F1 and F2 generations. For fatty acid content and total lipid yield, non-additive gene action was predominant in the F1 generation while additive gene action was predominant in the F2 generation under MWDS and SWDS conditions. The P4 parent had the highest general combining ability for fruit yield components in both the F1 and F2 generations. The P6 parent had the highest general combining ability for phenological and phytochemical traits. The P4 and P6 parents are promising material to develop early flowering and early maturing genotypes coupled with high total lipids in advanced generations of segregation.

Data mining for miRNAs and their targets in the Triticeae.

MicroRNAs (miRNAs) and the mRNA targets of miRNAs were identified by sequence complementarity within a DNA sequence database for species of the Triticeae. Data screening identified 28 miRNA precursor sequences from 15 miRNA families that contained conserved mature miRNA sequences within predicted stem-loop structures. In addition, the identification of 337 target sequences among Triticeae genes provided further evidence of the existence of 26 miRNA families in the cereals. MicroRNA targets included genes that are homologous to known targets in diverse model species as well as novel targets. MicroRNA precursors and targets were identified in 10 related species, though the great majority of them were identified in bread wheat, Triticum aestivum, and barley, Hordeum vulgare, the two species with the largest EST data sets among the Triticeae.

The alpha-tubulin gene family in wheat (Triticum aestivum L.) and differential gene expression during cold acclimation.

The alpha-tubulins and beta-tubulins are the major constituents of microtubules, which have been recognized as important structural elements in cell growth and morphogenesis, and, recently, for their role in regulation and signal transduction. We have identified 15 full-length cDNAs for the members of the alpha-tubulin gene family in hexaploid bread wheat (Triticum aestivum L.). The genes were clustered into 5 homeologous groups of 3 genes. Representatives of the 5 homeologous groups were mapped to different chromosome arms, and the genome of origin was determined for each gene. Changes in mRNA levels were observed for the paralogous members of the gene family during cold acclimation. Three members of the family had initial decreases in mRNA levels in response to cold treatment, which were followed by increases, each with a different pattern of reinduction. One gene-family member showed increased mRNA for up to 14 d during cold acclimation and had decreased levels after 36 d of cold treatment; a fifth paralogous member of the gene family had slowly declining mRNA levels up to 36 d. Subtle differences in the level of gene expression among homeologs and large differences among paralogs were detected by comparing the relative abundance of wheat alpha-tubulin expressed sequence tags (ESTs) in public databases.

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.

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.