Chromosome mapping of low-temperature induced Wcs120 family genes and regulation of cold-tolerance expression in wheat

 Low-temperature (LT) induced genes of the Wcs120 family in wheat (Triticum aestivum) were mapped to specific chromosome arms using Western and Southern blot analysis on the ditelocentric series in the cultivar Chinese Spring (CS). Identified genes were located on the long arms of the homoeologous group 6 chromosomes of all 3 genomes (A, B, and D) of hexaploid wheat. Related species carrying either the A, D, or AB genomes were also examined using Southern and Western analysis with the Wcs120 probe and the WCS120 antibody. All closely related species carrying one or more of the genomes of hexaploid wheat produced a 50 kDa protein that was identified by the antibody, and a Wcs120 homoeologue was detected by Southern analysis in all species. In the absence of chromosome arm 6DL in hexaploid CS wheat no 50 kDa protein was produced and the high-intensity Wcs120 band was missing, indicating 6DL as the location of Wcs120 but suggesting silencing of the Wcs120 homoeologue in the A genome. Levels of proteins that cross-reacted with the Wcs120 antibody and degrees of cold tolerance were also investigated in the Chinese Spring/Cheyenne (CS/CNN) chromosome substitution series. CNN chromosome 5A increased the cold tolerance of CS wheat. Densitometry scanning of Western blots to determine protein levels showed that the group 5 chromosome 5A had a regulatory effect on the expression of the Wcs120 gene family located on the group 6 chromosomes of all three hexaploid wheat genomes. Received: 10 July 1996 / Accepted: 30 September 1996     

Expression of the cold-induced wheat gene Wcs120 and its homologs in related species and interspecific combinations.

Low-temperature response was measured at the whole plant and at the molecular level in wheat-rye amphiploids and in other interspecific combinations. Cold tolerance of interspecifics whose parents diverged widely in hardiness levels resembled the less hardy higher ploidy level wheat parent. Expression of the low-temperature induced Wcs120 gene of wheat (Triticum aestivum L. em. Thell.) has been associated with freezing tolerance and was used here to study mRNA and protein accumulation in interspecific and parental lines during cold acclimation. Northern and Western analyses showed that homologous mRNAs and proteins were present in all the related species used in the experiments. Cold-tolerant rye (Secale cereale L.) produced a strong mRNA signal that was sustained throughout the entire 49-day cold-acclimation period. The wheats produced a mRNA signal that had diminished after 49 days of low-temperature exposure. The wheat-rye triticales did not exhibit the independent accumulation kinetics of the cold-tolerant rye parent but, rather, more closely resembled the wheat parent in that the mRNA signal was greatly diminished after 49 days of low-temperature exposure. The influence of the rye genome was manifest in slightly greater mRNA and protein accumulation in earlier stages of acclimation. Protein accumulations in the triticales were also maintained to a somewhat greater extent than found in the wheats at the end of the 49-day acclimation period. Protein accumulations in the wheat-crested wheatgrass (Agropyron cristatum L. Gaertner) interspecific resembled that of the wheat parent. The influence of the higher ploidy level wheats of the expression of homologous gene families from wheat-related hardy diploids in interspecific combinations may in part explain the poor cold tolerance observed.     

Physical mapping of wheat aquaporin genes

Aquaporins are water channel proteins that control the flow of water across cellular membranes and play vital roles in all aspects of plant–water relations. Our previous identification of 35 wheat PIP and TIP aquaporin genes showed they formed a large family with many conserved features that are thought to be important in structure and function. The present work focussed on determining the positions of these genes in the wheat genome in order to help investigate their functions in water uptake and transport. Genomic locations of wheat PIPs and TIPs were predicted using a number of reported rice–wheat comparative maps and additional in silico approaches. Physical mapping of select genes utilising aneuploid stocks and progenitor DNAs placed these on chromosomes 2B, 2D, 6B and 7B and helped to clarify the individual genes and homoeologues. The compilation of all in silico and physical mapping work confirmed many of the orthologous relationships between wheat and rice and/or barley genes, and synteny in the related areas of genome. These results further reinforce that wheat PIP and TIP proteins are most likely to have similar functions to those closely related in rice, including water permeability and abiotic stress response, and provide important tools for future investigations into the involvement of this complex gene family in traits related to plant-water relations and osmotic stress response.     

Genomic mapping of defense response genes in wheat

 Defense response (DR) genes are a broad class involved in plant defense. In this study we mapped 36 probes representing seven classes of defense response genes. This collection of probes represents genes involved in the hypersensitive response (HR), pathogenesis-related (PR) genes, genes for the flavonoid metabolic pathway, genes encoding proline/glycine-rich proteins, ion channel regulators, lipoxygenase, lectin, and others. Using nullisomic-tetrasomic lines of ‘Chinese Spring’, we were able to assign at least 167 loci to the 21 chromosomes of wheat. Homoeologous group 7 chromosomes possessed the most DR loci followed by group 2. Sixty-two loci were placed on existing genetic linkage maps of wheat. Map locations indicated that the DR gene loci are not randomly distributed throughout the wheat genome, but rather are located in clusters and/or in distal gene-rich regions of the chromosomes. Knowledge of the chromosomal locations and genome organization of DR genes will be useful for candidate gene analysis of quantitative trait loci. Received: 12 June 1998 / Accepted: 24 July 1998     

Chromosome assignment of eight SOX family genes in chicken

Chromosome locations of the eight SOX family genes, SOX1, SOX2, SOX3, SOX5, SOX9, SOX10, SOX14 and SOX21, were determined in the chicken by fluorescence in situ hybridization. The SOX1 and SOX21 genes were localized to chicken chromosome 1q3.1-->q3.2, SOX5 to chromosome 1p1.6-->p1.4, SOX10 to chromosome 1p1.6, and SOX3 to chromosome 4p1.2-->p1.1. The SOX2 and SOX14 genes were shown to be linked to chromosome 9 using two-colored FISH and chromosome painting, and the SOX9 gene was assigned to a pair of microchromosomes. These results suggest that these SOX genes form at least three clusters on chicken chromosomes. The seven SOX genes, SOX1, SOX2, SOX3, SOX5, SOX10, SOX14 and SOX21 were localized to chromosome segments with homologies to human chromosomes, indicating that the chromosome locations of SOX family genes are highly conserved between chicken and human.     

Low temperature-stimulated phosphorylation regulates the binding of nuclear factors to the promoter of Wcs120, a cold-specific gene in wheat

The Wcs120 gene encodes a highly abundant protein which appears to play an important role during cold acclimation of wheat. To understand the regulatory mechanism controlling its expression at low temperature, the promoter region has been characterized. Electrophoretic mobility shift assays using short promoter fragments revealed the presence in nuclear extracts from non-acclimated (NA) plants of multiple DNA-binding proteins which interact with several elements. In contrast, no DNA-binding activity was observed in the nuclear extracts from cold-acclimated (CA) plants. In vitro dephosphorylation of these CA nuclear extracts with alkaline phosphatase restored the binding activity. Moreover, okadaic acid (a potent phosphatase inhibitor) markedly stimulated the in vivo accumulation of the WCS120 family of proteins. This suggests that protein phosphatases PP1 and/or PP2A negatively regulate the expression of the Wcs120 gene. In addition, both Ca²⁺-dependent and Ca²⁺-independent kinase activities were found to be significantly higher in the CA nuclear extracts. Western analysis using antibodies directed against protein kinase C (PKC) isoforms showed that a PKCγ homolog (84?kDa) is selectively translocated into the nucleus in response to low temperature. Taken together, our results suggest that, in vivo, the expression of the Wcs120 gene may be regulated by nuclear factors whose binding activity is modulated by a phosphorylation/dephosphorylation mechanism.     

Chromosome mapping and identification of amphiphilic proteins of hexaploid wheat kernels

Amphiphilic proteomic analysis was carried out on the ITMI (International Triticae Mapping Population) population resulting from a cross between "Synthetic", i.e.: "W7984" and "Opata". Out of a total of 446 spots, 170 were specific to either of the two parents, and 276 were common to both. Preliminary analysis, which was performed on 80 progenies (Amiour et al. 2002a), was completed here using a total of 101 selfed lines. Seventy two Loci of amphiphilic spots placed at LOD = 5 were conclusively assigned to15 chromosomes. Some spots mapped during the first analysis were eliminated because of the significant distortion segregation observed in the second analysis. Group-1 chromosomes had by far the greatest number of mapped spots (51). Using the Quantitative Trait Loci (QTLs) approach, analysis of the quantitative variation of each spot revealed that 96 spots out of the 170 specific ones showed at least one Protein Quantity Locus (PQL). These PQLs were distributed throughout the genome. With Matrix Laser Desorption Ionisation Time Of Flight (MALDI-TOF) spectrometry and Database interrogation, a total of 93 specific and 41 common spots were identified. This enabled us to show that the majority of these proteins are associated with membranes and/or play a role in plant defence against external invasions. Using multiple-regression analysis, other amphiphilic proteins, in addition to puroindolines, were shown to be involved in variation in kernel hardness in the ITMI population.Communicated by J.W. Snape     

Molecular mapping of gibberellin-responsive dwarfing genes in bread wheat

Opportunities exist for replacing reduced height (Rht) genes Rht-B1b and Rht-D1b with alternative dwarfing genes for bread wheat improvement. In this study, the chromosomal locations of several height-reducing genes were determined by screening populations of recombinant inbred lines or doubled haploid lines varying for plant height with microsatellite markers. Linked markers were found for Rht5 (on chromosome 3BS), Rht12 (5AL) and Rht13 (7BS), which accounted for most of the phenotypic variance in height in the respective populations. Large height differences between genotypes (up to 43 cm) indicated linkage to major height-reducing genes. Rht4 was associated with molecular markers on chromosome 2BL, accounting for up to 30% of the variance in height. Confirming previous studies, Rht8 was linked to markers on chromosome 2DS, whereas a population varying for Rht9 revealed a region with a small but significant height effect on chromosome 5AL. The height-reducing effect of these dwarfing genes was repeatable across a range of environments. The molecular markers developed in this study will be useful for marker-assisted selection of alternative height-reducing genes, and to better understand the effects of different Rht genes on wheat growth and agronomic performance.     

Activity-dependent regulation of BRINP family genes

We previously identified a family of novel developmentally regulated genes: BRINP1, 2, and 3, which are predominantly and widely expressed in the CNS from earlier developmental stages to adulthood. In the present study, we investigated the activity-dependent regulation of BRINP expression in the CNS. Among the three BRINP genes, BRINP1-mRNA was specifically up-regulated in the dentate gyrus of mouse hippocampus by kainic acid treatment. In cultured hippocampal neurons, the induction of BRINP1-mRNA was also observed by the activation of glutamate receptors. Although BDNF-mRNA is up-regulated in a similar activity-dependent manner, BDNF itself did not induce BRINP1-mRNA. From these results, the physiological roles of the activity-dependent induction of BRINP1-mRNA are discussed.     

Chromosome assignment of four photosynthesis-related genes and their variability in wheat species

Copy numbers of four photosynthesis-related genes, PhyA, Ppc, RbcS and Lhcb1 ^*1, in wheat genomes were estimated by slot-blot analysis, and these genes were assigned to the chromosome arms of common wheat by Southern hybridization of DNA from an aneuploid series of the cultivar Chinese Spring. The copy number of PhyA was estimated to be one locus per haploid genome, and this gene was assigned to chromosomes 4AL, 4BS and 4DS. The Ppc gene showed a low copy number of small multigenes, and was located on the short arm of homoeologous group 3 chromosomes and the long arm of chromosomes of homoeologous group 7. RbcS consisted of a multigene family, with approximately 100 copies in the common wheat genome, and was located on the short arm of group 2 chromosomes and the long arm of group 5 chromosomes. Lhcb1 ^*1 also consisted of a multigene family with about 50 copies in common wheat. Only a limited number of restriction fragments (approximately 15%) were used to determine the locations of members of this family on the long arm of group 1 chromosomes owing to the multiplicity of DNA bands. The variability of hybridized bands with the four genes was less in polyploids, but was more in the case of multigene families. RFLP analysis of polyploid wheats and their presumed ancestors was carried out with probes of the oat PhyA gene, the maize Ppc gene, the wheat RbcS gene and the wheat Lhcb1 ^*1 gene. The RFLP patterns of common wheat most closely resembled those of T. Dicoccum (Emmer wheat), T. urartu (A genome), Ae. speltoides (S genome) and Ae. squarrosa (D genome). Diversification of genes in the wheat complex appear to have occurred mainly at the diploid level. Based on RFLP patterns, B and S genomes were clustered into two major groups. The fragment numbers per genome were reduced in proportion to the increase of ploidy level for all four genes, suggesting that some mechanism(s) might operate to restrict, and so keep to a minimum, the gene numbers in the polyploid genomes. However, the RbcS genes, located on 2BS, were more conserved (double dosage), indicating that the above mechanism(s) does not operate equally on individual genes.     

Chromosome mapping and phylogenetic analysis of the cytosolic acetyl-CoA carboxylase loci in wheat

The cytosolic isoform of plant acetyl-CoA carboxylase is a multidomain enzyme involved in the synthesis of very-long-chain fatty acids and in secondary metabolism. Chromosome mapping of wheat identified one locus containing cytosolic acetyl-CoA carboxylase genes (Acc-2) and a related partially processed pseudogene (Psi-Acc-2) in the distal region of the long arm of wheat homoeologous group 3 chromosomes. Multiple copies of the Acc-2 genes, whose presence was suggested by sequence analysis, are likely to be arranged in tandem repeats. At least three out of five genes cloned from hexaploid wheat map to this locus. Another locus containing Acc-2--related sequences is present in the distal region of the long arm of chromosome 5D. The identity of the hybridizing DNA present at this locus remains unknown. A system based on PCR-cloning and DNA sequence analysis of acetyl-CoA carboxylase genes was developed to address various phylogenetic and systematics questions in grasses. It was applied to reconstruct the phylogeny of the Acc-2 genes from D- and S-genome Aegilops and A-genome Triticum diploid species, AABB- and AAGG-genome tetraploid wheat, and AABBDD-genome hexaploid wheat, as well as from rye and barley. The combined cytogenetic and molecular evolution approach allowed assignment of gene sequences included in phylogenetic analysis to specific loci on homoeologous chromosomes. Recurring gene duplication followed by chromosome translocation and/or possible loss of some gene copies, as well as loss of introns, occurred in the gene family in different plant lineages. Two major Acc-2 clades appeared before the divergence of barley and rye. Nucleotide substitution rates in different parts of the Acc-2 gene were assessed. This analysis of the Acc-2 loci provides detailed information regarding evolutionary events at a low--copy-number locus containing important functional genes. These events are likely to be common and to play a significant role in shaping grass genomes.     

Linkage mapping of genes controlling endosperm storage proteins in wheat

Summary A translocation mapping procedure was used to map gene-centromere distances for the genes controlling endosperm proteins on the short arm of each of the chromosomes 1A, 1B and 1D in wheat. The genes controlling triplet proteins (tentatively designated Tri-1) were found to be closely linked to the centromere on chromosome arms 1AS and 1DS and loosely linked to the gliadin genes (Gli-1) on the same arms. The Gli-1 genes segregated independently or were very loosely linked to their respective centromeres. The Gli-B1-centromere map distance on 1BS was also estimated using conventional telocentric mapping and the result was similar to that obtained with the translocation mapping. A simple two-step one-dimensional electrophoretic procedure is described which allows the low-molecular-weight (LMW) glutenin subunits to be separated from the gliadin bands, thus facilitating the genetic analysis of these LMW subunits. No recombination was observed between the genes (designated Glu-3) controlling some major LMW glutenin subunits and those controlling gliadins on chromosome arms 1AS and 1DS. However, in a separate experiment, the genes controlling LMW glutenin subunits on 1BS (Glu-B3) showed a low frequency of recombination with the gliadin genes.Portion of the Ph.D. thesis submitted by the senior author     

WCS120 protein family and proteins soluble upon boiling in cold-acclimated winter wheat

The amount of proteins soluble upon boiling (especially WCS120 proteins) and the ability to develop frost tolerance (FT) after cold acclimation was studied in two frost-tolerant winter wheat cultivars, Mironovskaya 808 and Bezostaya 1. Protein gel blot analysis, mass spectrometry (MS) and image analysis of two-dimensional gel electrophoresis (2-DE) gels were used to identify and/or quantify the differences in protein patterns before (non-acclimated, NA) and after 3 weeks of cold acclimation (CA) of the wheats, when FT increased from -4 degrees C (lethal temperature (LT(50)), for both cultivars) to -18.6 degrees C in Bezostaya 1 and -20.8 degrees C in Mironovskaya 808. Only WCS120 protein was visible in NA leaves while all five WCS120 proteins were induced in the CA leaves. Mironovskaya 808 had higher accumulation of three members of WCS120 proteins (WCS120, WCS66 and WCS40) than Bezostaya 1. MS analysis of total sample of proteins soluble upon boiling showed seven COR proteins in the CA samples and only three COR proteins in the NA samples of cultivar Mironovskaya 808 (MIR). In conclusion, the level of the accumulation of WCS120, WCS66 and WCS40 distinguished our two frost-tolerant winter wheat cultivars. Moreover, the differences of CA and NA samples of the MIR were shown by liquid chromatography (LC)-tandem mass spectrometry (MS/MS).     

Intrachromosomal mapping of crossability genes in wheat (Triticum aestivum)

Summary Intrachromosomal mapping studies were used to locate the positions of the genes Kr1 and Kr2, which control the crossability of wheat with Hordeum bulbosum, on chromosomes 5B and 5A, respectively. The location of Kr1 was established using the telocentric mapping technique and found to be on the long arm of chromosome 5B, distal to the centromere with a mean recombination frequency of 44.8±3.28%. Kr2 was located on the long arm of chromosome 5A by linkage with the major gene markers Vrn1, controlling vernalization requirement, and q, controlling ear morphology. Kr2 is closely linked to Vrn1, with a mean recombination frequency of 4.8±4.66%, and is distal to q with a mean recombination frequency of 38.1±10.60%. The similar locations of Kr1 and Kr2 on homoeologous chromosomes suggest that these two loci are homoeoallelic. Significant correlations between Hordeum bulbosum and rye crossability confirmed that Kr1 and Kr2 control the crossability of wheat with both species.     

Galectin genes: regulation of expression

In this review we have summarized the more recent studies on the expression of mammalian galectins. One interesting observation that can be made is that in most of microarrays and/or differential display analysis performed in recent years one or more galectins have been picked up. From a critical evaluation of the pertinent studies the main conclusion that can be drawn is that, although it is not yet clear whether the 14 galectins identified so far have functions in common, a striking common feature of all galectins is the strong modulation of their expression during development, differentiation stages and under different physiological or pathological conditions. This suggests that the expression of different galectins is finely tuned and possibly coordinated. In spite of these observations it is rather unexpected that very few studies have been performed on the molecular mechanisms governing the activity of galectin genes.     

Chromosome localization of human ARH genes, a ras-related gene family

The human ARH genes (previously called RHO) share several properties with the ras gene family. Three members of the ARH family, the H6, H9, and H12 genes, have been localized to human chromosomes 2, 5, and 3, respectively. Analysis of DNAs from a rodent-human somatic cell hybrid panel demonstrates linkage of H6 to chromosome region 2p12----2pter and H9 to region 5q33----5qter. In situ chromosome hybridization also showed that the primary site for H9 is in the 5q31----qter region. The H12 gene was some-what difficult to localize using rodent-human hybrids because the probe detects a family of rodent genes as homologous to the human probe as in the human cognate gene. However, chromosome in situ hybridization revealed grains clustered in region 3p14----3p22 with a significant peak in band 3p21. We conclude that H6 is in 2p12----pter, H9 in 5q31----5qter, and H12 in 3p21.